Methods of inducing immune tolerance using immunotoxins

ABSTRACT

Provided is a method of treating an immune system disorder not involving T cell proliferation, comprising administering to the animal an immunotoxin comprising a mutant diphtheria toxin moiety linked to an antibody moiety which routes by the anti-CD3 pathway, or derivatives thereof under conditions such that the disorder is treated. Thus, the present method can treat graft-versus-host disease. Also provided is a method of inhibiting a rejection response by inducing immune tolerance in a recipient to a foreign mammalian donor tissue or cells, comprising the steps of: a) exposing the recipient to an immunotoxin so as to reduce the recipients&#39;s peripheral blood T-cell lymphocyte population by at least 80%, wherein the immunotoxin is anti-CD3 antibody linked to a diphtheria protein toxin, wherein the protein has a binding site mutation; and b) transplanting the donor cells into the recipient, whereby a rejection response by the recipient to the donor organ cell is inhibited, and the host is tolerized to the donor cell.

This application is a continuation of application No. 08/843,409, filedApr. 15, 1997, now U.S. Pat. No. 6,103,235, which is incorporated hereinby reference in its entirety and which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/015,459, filed Apr. 15, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to an immunotoxin and to techniques forinducing immunological tolerance in primates. It appears to beespecially well suited to provide a method for inhibiting rejection oftransplanted organs. The invention further relates to a method oftreating T cell leukemias or lymphomas, graft-versus-host diseases, andautoimmune diseases by administering an immunotoxin.

2. Background Art

The number of organ transplants performed in the United States isapproximately 19,000 annually and consists predominantly of kidneytransplants (11,000), liver transplants (3,600), heart transplants(2,300), and smaller numbers of pancreas, lung, heart-lung, andintestinal transplants. Since 1989 when the United Network for OrganSharing began keeping national statistics, approximately 190,000 organtransplants have been performed in the United States. A large butdifficult to ascertain number of transplants were performed in theUnited States prior to 1989 and a similarly large number of transplantsare performed in Europe and Australia and a smaller number in Asia.

Transplant tolerance remains an elusive goal for patients and physicianswhose ideal would be to see a successful, allogeneic organ transplantperformed without the need for indefinite, non-specific maintenanceimmunosuppressive drugs and their attendant side effects. Over the past10 years the majority of these patients have been treated withcyclosporin, azathioprine, and prednisone with a variety of otherimmunosuppressive agents being used as well for either induction ormaintenance immunosuppression. The average annual cost of maintenanceimmunosuppressive therapy in the United States is approximately $10,000.While the efficacy of these agents in preventing rejection is good, theside effects of immunosuppressive therapy are considerable because theunresponsiveness which they induce is nonspecific. For example,recipients can become very susceptible to infection. A major goal intransplant immunobiology is the development of specific immunologictolerance to organ transplants with the potential of freeing patientsfrom the side effects of continuous pharmacologic immunosuppression andits attendant complications and costs.

Anti-T cell therapy (anti-lymphocyte globulin) has been used in rodentsin conjunction with thymic injection of donor cells (Posselt et al.Science 1990; 249: 1293-1295 and Remuzzi et al. Lancet 1991; 337:750-752). Thymic tolerance has proved successful in rodent models andinvolves the exposure of the recipient thymus gland to donor alloantigenprior to an organ allograft from the same donor. However, thymictolerance has never been demonstrated in large animals, and itsrelevance to tolerance-in humans in unknown.

One approach to try to achieve such immunosuppression has been to exposethe recipient to cells from the donor prior to the transplant, with thehope of inducing tolerance to a later transplant. This approach hasinvolved placement of donor cells (e.g. bone marrow) presenting MHCClass I antigens in the recipient's thymus shortly after application ofanti-lymphocyte serum (ALS) or radiation. However, this approach hasproved difficult to adapt to live primates (e.g. monkeys; humans). ALSand/or radiation render the host susceptible to disease or side-effectsand/or are insufficiently effective.

If a reliable, safe approach to specific immunologic tolerance could bedeveloped, this would be of tremendous value and appeal to patients andtransplant physicians throughout the world with immediate application tonew organ transplants and with potential application to transplantrecipients with stable function. Thus, a highly specificimmunosuppression is desired. Furthermore, there is a need for a meansfor imparting tolerance in primates, without the adverse attributes ofusing ALS or radiation. Moreover, the goal is to achieve more thansimply delaying the rejection response. Rather, an important goal is toinhibit the rejection response to the point that rejection is not afactor in reducing average life span.

The present invention meets this need by providing a method of inducingimmune tolerance.

SUMMARY OF THE INVENTION

It is an object of this invention to provide an immunotoxin for treatingimmune system disorders.

It is a further object of the invention to provide a method of treatingan immune system disorder not involving T cell proliferation, comprisingadministering to the animal an immunotoxin comprising a mutantdiphtheria toxin moiety linked to an antibody moiety which routes by theanti-CD3 pathway, or derivatives thereof under conditions such that thedisorder is treated. Thus, the present method can treatgraft-versus-host disease.

It is a further object of the invention to provide a method of inducingimmune tolerance. Thus, the invention provides a method of inhibiting arejection response by inducing immune tolerance in a recipient to aforeign mammalian donor tissue or cells, comprising the steps of: a)exposing the recipient to an immunotoxin so as to reduce therecipients's peripheral blood T-cell lymphocyte population by at least80%, wherein the immunotoxin is anti-CD3 antibody linked to a diphtheriaprotein toxin, wherein the protein has a binding site mutation; and b)transplanting the donor cells into the recipient, whereby a rejectionresponse by the recipient to the donor organ cell is inhibited, and thehost is tolerized to the donor cell.

The objects of the invention therefore include providing methods of theabove kind for inducing tolerance to transplanted organs or cells fromthose organs. This and still other objects and advantages of the presentinvention will be apparent from the description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows nude mice bg/nu/xid maintained in a semi-sterileenvironment are preconditioned with 400 cGy whole body ¹³⁷CS γ radiationon day −7. On day 0, 2.5×10⁷ Jurkat cells (human T cell leukemia CD3+,CD4+, CD5+) are injected subcutaneously with 1×10⁷ HT-1080 feeder cells(human sarcoma) which have received 6000 cGy. Jurkat cells were passagedevery other week in mice as subcutaneous tumors and dissociated bycollagenase/dispase prior to inoculation. This cell population exhibitsa 40% inhibition of protein synthesis after 5 hours exposure to 10¹¹Manti-CD3-DT. Clones isolated from this population by infinite dilutionexhibit varying sensitivity to anti-CD3-DT (4 less sensitive, 3 moresensitive) corresponding to a 1.5 log variation in dose response curves.Immunotoxin treatment is given by intraperitoneal injection starting onday 7 when the tumor is visibly established. Evaluation takes place onday 37.

FIG. 2 shows that the epitopes involved in human serum's inhibition oftoxicity lie in the last 150 amino acids of DT. A schematic diagram ofthe DT mutants CRM9, CRM197 and MSPΔ5 is presented (A). The A- andB-subfragments and their relative size and position are shown. Thefilled circle represents a point mutation as described in the text. Goat(B) or human (C) serum )human serum was a pool from all samples withpositive ELISA for anti-DT antibodies) was incubated with increasingmolar concentrations of CRM197 (—O—), MSPΔ5 (—X—) or the B-subfragment(—Δ—) of DT for 30 minutes at room temperature. To this reaction,UCHT1-CRM9 was added to a final concentration of 1×10⁻¹⁰ M. This mixturewas then diluted 10-fold onto Jurkat cells in a protein synthesisinhibition assay as described in the Materials and Methods. Immunotoxinincubated with medium only inhibited protein synthesis to 4% ofcontrols. The results are representative of two independent assays.

FIG. 3 shows that sFv-DT390 maintains specificity for the CD3 complexbut is 16-fold less toxic than UCHT1-CRM9 to Jurkat cells. A) Increasingconcentrations of sFv-DT390 (—Δ—) or UCHT1-CRM9 (—O—) were tested inprotein synthesis inhibition assays as described in the Materials andMethods. The results are an average of four separate experiments. B)Increasing-concentrations of UCHT1 antibody were mixed with a 1×10⁻¹⁰ MUCHT1-CRM9 (—O—) or 3.3×10⁻¹⁰ M sFv-DT390 (—Δ—) and then added to cellsfor a protein synthesis inhibition assay.

FIG. 4 shows the schematic flow sheet for generation of the single chainantibody scUCHT1 gene construct. PCR: polymerase chain reaction; L:linker; SP: signal peptide. P1 to P6, SP1, and SP2 are primers used inPCR, and listed in table 1.

FIG. 5 shows the western blotting analysis of the single chain antibodyscUCHT1. scUCHT1 was immunoprecipitated, and separated on 4-20% SDS/PAGEgradient gel. After transferring to Problott™ membrane, scUCHT1 wasvisualized by an anti-human IgM antibody labeled with phosphatase.scUCHT1 secreted was mainly a dimeric form. Lane 1-3 representingelectrophoresis under reducing conditions, and 4-6 non-reducingconditions. Lane 1 and 6 are human IgM; lane 1: IgM heavy chain. Thelight chain is not visible, because the anti-IgM antibody is directed atthe heavy chain; lane 6: IgM pentamer is shown as indicated by thearrow. Lane.2 and 4 scUCHT1 from COS-7 cells; 3 and 5 scUCHT1 from SP2/0cells.

FIG. 6 shows that scUCHT1 had the same specificity and affinity as itsparental antibody UCHT1. In the competition assay, ¹²⁵I-UCHT1 was usedas tracer in binding Jurkat cells. scUCHT1 from COS-7 (□) and SP2/0cells (Δ), or unlabeled UCHT1 (∘) with indicated concentrations wereincluded as competitor. Results were expressed as a percentage of the¹²⁵I-UCHT1 bound to cells in the absence of competitors.

FIG. 7 shows that scUCHT1 did not induce human T cell proliferationresponse. scUCHT1 from COS-7 (Δ) and SP2/0 (∘) cells and UCHT1 (□) wereadded to human PBMCs at indicated concentrations and T cellproliferation was assayed by [3H]thymidine incorporation. UCHT1 induceda vigorous proliferation response. On the contrary, scUCHT1 had littleeffect at any doses.

FIG. 8 a shows that UCHT1 and scUCHT1 had little effect on TNF-αsecretion, and. scUCHT1 from both COS-7 (Δ) and SP2/0 (∘) cells andUCHT1 (□) were added to cultures of human blood mononuclear cells.Culture supernatant was harvested and used for ELISA determination ofTNF-α and IFN-γ as described in materials and methods.

FIG. 8 b shows that UCHT1 and scUCHT1 inhibited the basal production ofIFN-γ. scUCHT1 from both COS-7 (Δ) and SP2/0 (∘) cells and UCHT1 (□)were added to cultures of human blood mononuclear cells. Culturesupernatant was harvested and used for ELISA determination of TNF-α andIFN-γ as described in materials and methods.

FIG. 9 is a western blot showing the secreted scUCHT1 immunotoxin.

FIG. 10 shows a PCR amplification scheme.

FIG. 11 shows one clone expressing the divalent immunotoxin fusionprotein shown in FIG. 13.

FIG. 12 shows another clone expressing a divalent immunotoxin fusionprotein shown in FIG. 14.

FIG. 13 is a schematic of a divalent fusion immunotoxin.

FIG. 14 is a schematic of a divalent fusion immunotoxin.

FIG. 15 is a schematic of a divalent fusion immunotoxin.

FIG. 16 shows the cloning scheme used to obtain scUCHT1 fusion proteinwith DTM1 and DT 483.

FIG. 17 shows the cloning scheme used to obtain scUCHT1 fusion proteinwith DT 390.

FIG. 18 shows the cloning scheme used to obtain scUCHT1 fusion proteinwith DT 370.

FIG. 19 a shows CD3+ cell depletion and recovery in peripheral bloodfollowing immunotoxin treatment. Days refer to days after the first doseof immunotoxin.

FIG. 19 b shows CD3+ cell depletion in lymph nodes following immunotoxintreatment.

FIG. 20 shows the rise in serum IL-12 following FN18-CRM9 immunotoxintreatment in post kidney transplant monkeys with and without treatmentwith DSG (deoxyspergualin) and solumedrol.

FIG. 21 shows the rise in serum IFN-gamma following FN18-CRM9immunotoxin treatment in post kidney transplant monkeys with and withouttreatment with DSG and solumedrol. The treatment dramatically attenuatesthe rise of IFN-gamma.

FIG. 22 shows that DSG and solumedrol treatment in the peritransplantperiod following immunotoxin suppresses weight gain, a sign of vascularleak syndrome related to IFN-gamma elevation.

FIG. 23 shows that DSG and solumedrol treatment in the peritransplantperiod following immunotoxin suppresses hypoproteinemia, a sign ofvascular leak syndrome related to IFN-gamma elevation.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides immunotoxins and methods of using them to induceimmune tolerance and to treat disease.

Immunotoxin.

The present invention relates to an immunotoxin. More specifically, animmunotoxin, comprising a mutant diphtheria toxin moiety linked to asingle chain variable region antibody which routes by the anti-CD3pathway is provided. The immunotoxin can be divalent. The immunotoxincan be a fusion protein produced recombinantly. The antibody moiety ofthe immunotoxin can comprise the human CH2 and CH3 regions. Theseregions can be from the antibody UCHT1 so that the antibody moiety isscUCHT1, which is a single chain CD3 antibody having human CH2 and CH3regions and mouse variable regions as shown in the figures. These arethe first instances of a sc anti-CD3 antibodies. Numerous DT mutanttoxin moieties are described herein, for example DT390. Thus, as justone specific example the immunotoxin, the invention providesscUCHT1-DT390. Derivatives of this immunotoxin are designed andconstructed as described herein.

The toxin moiety retains its toxic function, and membrane translocationfunction to the cytosol in full amounts. The loss in binding functionlocated in the C terminus of the protein diminishes systemic toxicity byreducing binding to non-target cells. Thus, the immunotoxin can besafely administered. The routing function normally supplied by the toxinbinding function is supplied by the targeting antibody anti-CD3. Theessential routing pathway is (1) localization to coated pits forendocytosis, (2) escape from lysosomal routing, and (3) return to theplasma membrane. Any antibody which can route in this manner will beeffective with the toxin moiety, irrespective of the epitope to whichthe antibody is directed. Thus, a wide variety of cell types can inprinciple be targeted. When antibodies dissociate from their receptorsdue to changes in receptor configuration induced in certain receptors asa consequence of endosomal acidification, they enter the lysosomalpathway. This can be prevented or minimized by directing the antibodytowards an ecto-domain epitope on the same receptor which is closer tothe plasma membranes (Ruud, et al. (1989) Scand. J. Immunol. 29:299;Herz et al. (1990) J. Biol. Chem. 265:21355). Other DT binding sitemutants can be used to form derivatives by changing amino acids in theC-terminus which can reduce the binding function as long as thetranslocation function is maintained. Specific examples are described inthe Examples.

In another embodiment, the present invention relates to an anti-CD3-CRM9immunotoxin or derivatives thereof. The design of successful derivativesof anti-CD3-CRM9 depend upon understanding how the unique concentrationof anti-CD3-CRM9 achieves its biological effect.

An example of a series of derivatives which is likely to be effectiveare antibody-CRM9 conjugates directed at unique Vα and Vβ gene segmentproducts of the T cell receptor. Some of these epitopes appear to bebiased towards specific autoimmune processes. Such conjugates should beuseful in specific autoimmune diseases (Kappler et al. (1987) Cell49:263; Urban et al. (1988) Cell 54:577).

Relatedly, the invention provides an anti-Vβ-CRM9 immunoconjugate suchas anti-Vβ₁₂-CRM9. Also provided is an anti-Vα-CRM9 immunoconjugate.Both of the conjugates can be placed in a pharmaceutically acceptablecarrier for administration to a subject. Both acid-cleavable andnon-cleavable protein cross-linking reagents can be used in theconstruction of antibody-diphtheria toxin binding-site mutant conjugateslike anti-CD3-CRM9 (Neville et al. (1989) J. Biol. Chem.264:14653-14661); preferred are non-cleavable crosslinkers, such asbismaleimidohexane and m-maleimidobenzoyl-N-hydroxysuccinimide ester.The synthesis of acid-cleavable protein cross-linking reagents based onorthoester, acetal, and ketal functionalities has been described(Srinivasachar and Neville (1989) Biochemistry 28:2501-2509). The uniquefeature of these functionalities is that their observed hydrolytic rateconstants increase 10-fold for each drop in pH, a consequence ofspecific H₃O° catalysis leading to a carbonium ion intermediate (Cordesand Bull (1974) Chem. Rev. 74:581-603). Moreover, these functionalitiesare resistant to base catalysis permitting manipulation and storage atalkaline pH. The cross-linking reagents react with proteins viaheterobifunctional groups (maleimide and N-hydroxysuccinimide ester) orhomobifunctional groups (bis-maleimide). The maleimide cross-linking isaccomplished by prior protein thiolation with iminothiolane.Cross-linked proteins exhibit first-order dissociation under acidconditions. The t_(1/2) at pH 5.5 varies between 0.1 and 130 h for aseries of six different cleavable cross-linkers (Srinivasachar andNeville (1989) Biochemistry 28:2501-2509).

The mutant diphtheria toxin moiety can be a truncated mutant, such asDT390, DT383, DT370 or other truncated mutants, as well as a full lengthtoxin with point mutations, such as DTM1, as described in Examples 9-11.scUCHT1 fusion proteins with DTM1 and DT483 (see FIG. 16), DT390 (FIG.17) and DT370 (FIG. 18) have been cloned and expressed in E. coli. Theantibody moiety can be scUCHT1 or other anti-CD3 antibody having thecharacteristics set forth herein. Thus, one example of an immunotoxinfor use in the present methods is UCHT1-DT390. The describedimmunotoxins can be used in all the methods of the invention.

Other examples of immunotoxins include anti-Vβ-CRM9 and anti-Vα-CRM9.For example, the antibody-CRM9 conjugate used in any of the methodsherein can be an anti-Vβ-CRM9 such as anti-Vβ₈-CRM9. In addition, theantibody-CRM9 conjugate can be an anti-Vα-CRM9. In one embodiment, theanti-Vβ-CRM9 is anti-Vβ₁₂-CRM9 and the disease is human immunodeficiencyvirus disease or the Acquired Immunodeficiency Syndrome (AIDS). Other Vαand Vβ targets associated with particular autoimmune diseases exist. Forexample, pulmonary sarcoidosis showed increased usage of the Vβ₈ subsetin blood and lung lymphocytes (Moller et al. (1988) J. Clin. Invest.82:1183-1191). In multiple sclerosis, preferential use of the Vβ_(5.2)subset in brain plaque lesions has been identified and rearrangements OfVα_(1,2,7,8,and 10) were also prominent (Oksenberg et al. (1993) Nature362:68-70).

The antibody-toxin constructs of the invention can be expected to beeffective as immunotoxins, because the relevant parameters are known.The following discussion of parameters is relevant to the use of theimmunotoxin in tolerance induction. The relevant binding constants,number of receptors and translocation rates for humans have beendetermined and used. Binding values for anti-CD3-CRM9 for targeted andnon-targeted cells in vitro are described above at page 2. Rates oftranslocation for the anti-CD3-CRM9 conjugate to targeted andnon-targeted cells in vitro are described in references cited at page 2(Greenfield et al. (1987) Science 238:536; Johnson et al. (1988) J.Biol. Chem. 263:1295; Johnson et al. (1989) J. Neurosurg. 70:240; andNeville et al. (1989) J. Biol. Chem. 264:14653). The rate limitingtranslocation rate to targeted cells in vitro is recited at page 5,wherein it is shown that the conjugate is translocated to about 40% ofthe target cells present as measured by inhibition of protein synthesisin about 40% of cells. Inhibition of protein synthesis is complete incells into which the conjugate translocates.

Parameters determined in in vivo studies in nude mice include thefollowing: Tumor burden is described in Example 1 as a constant massequal to 0.1% of body weight; the receptor number and variation ofreceptor number are described in Example 3; “favorable therapeuticmargin” is defined as an in vivo target cell 3 log kill at 0.5 MLD(minimum lethal dose) comparison of efficacy with an establishedtreatment of 0.5 MLD immunotoxin equivalent (group 1) to a radiationdose of 500-600 cGy (groups 8 and 9).

The parameters determined in vitro allowed the prediction of success inthe in vivo nude mouse study. The prediction of in vivo success wasverified by the data in Examples 3-4. Using the target cell number fromthe mouse study as being equivalent to the local T cell burden in amonkey or man successful T cell ablation and immunosuppression inmonkeys could be predicted. This prediction has been verified by themonkey data in Examples 5 and 7-8. Using the same parameters, ascientist skilled in this field can make a prediction of success inhumans with confidence, because these parameters have been previouslyshown to have predictive success.

In another embodiment, the present invention relates to a pharmaceuticalcomposition comprising anti-CD3-DT mutant in an amount effective totreat T cell leukemias or lymphomas which carry the CD3 epitope,graft-versus-host disease or autoimmune diseases, and a pharmaceuticallyacceptable diluent, carrier, or excipient. One skilled in the art willappreciate that the amounts to be administered for any particulartreatment protocol can readily be determined. Suitable amounts might beexpected to fall within the range of 0.01 to 1.0 mg (toxin content) perkg of body weight.

Non-toxic mutant of diphtheria toxin.

Most human sera contain anti-DT neutralizing antibodies from childhoodimmunization. To compensate for this the therapeutic dose ofanti-CD3-CRM9 can be appropriately raised without affecting thetherapeutic margin. Alternatively, the present application provides anon-toxic DT mutant reactive with neutralizing antisera (e.g., CRM197)that can be administered in conjunction with the immunotoxin.

A non-toxic mutant of diphtheria toxin for use in the present methodscan be DTM2 or CRM197. DTM2 and CRM197 are non-toxic mutants of DT,having a point mutation in the enzymatic chain. However, they have thefull antigenic properties of DT and CRM9, and CRM197 is used forimmunization (Barbour et al. 1993. Pediatr Infect. Dis. J. 12: 478-84).Other non-toxic DT mutants that can be used in the present method willshare the characteristic of totally lacking A chain enzymatic activity.

The purpose of administering the non-toxic toxin is to bind preexistinganti-CRM9 and anti-DT antibodies in a subject and compete with theireffect and/or induce their removal from the circulation. Thissubstantially avoids any host immune response to the immunotoxin thatmight interfere with the activity of the immunotoxin.

The protein synthesis inhibition assay in the presence of human serumsamples or pooled human sera described in the Examples becomes animportant part of the evaluation of the optimal immunotoxin for theindividual patient and is provide for this purpose. This assay makesroutine the systematic evaluation of additional combinations of DT pointmutations and carboxy-terminal deletions for the purpose of minimizingblockade of immunotoxin in vivo by anti-human antitoxin.

The non-toxic mutant is preferably administered concurrently with orshortly before the immunotoxin. For example, the non-toxic DT mutant canbe administered within an hour, and preferably about 5 minutes prior tothe administration of immunotoxin. A range of doses of the non-toxicmutant can be administered. For example, an approximately 10 to 100 foldexcess of non-toxic mutant over the CRM9 content of the immunotoxin tobe administered can be administered by I.V. route.

Another use of the non-toxic DT mutant in the present methods is to runthe recipient patient's blood through a column containing the non-toxicDT mutant to remove some or all of the patient's serum antibodiesagainst DT.

Method of Inducing Immune Tolerance.

One embodiment to the invention provides a method of inhibiting arejection response by inducing immune tolerance in a recipient to aforeign mammalian donor organ cell by safely exposing the recipient toan immunotoxin so as to reduce the recipients's peripheral blood T-celllymphocyte population by at least 80%, and preferably 95% or higher,wherein the immunotoxin is an anti-CD3 antibody linked to a diphtheriaprotein toxin, and wherein the protein has a binding site mutation. Theterm “safely” in this context means that recipient is not killed by theimmunotoxin. The term “donor cell” refers to a donor organ or a cell orcells of the donor organ, as distinguished from donor lymphocytes ordonor bone marrow. When the donor organ or cells of the donor istransplanted into the recipient, a rejection response by the recipientto the donor organ cell is inhibited and the recipient is tolerized tothe donor organ cell. Alternatively, a non-toxic DT mutant such as DTM2or CRM197 can first be administered followed by the immunotoxin. Thismethod can use any of the immunotoxins (e.g., anti-CD3-CRM9,scUCHT1-DT390, etc.) or non-toxic DT mutants described herein with thedosages and modes of administration as described herein or otherwisedetermined by the practitioner.

As further described in the Examples, the above-described method forinducing tolerance can be augmented by additional treatment regimens.For example, the method can further include administering to the thymusgland a thymic apoptosis signal before, at the same time, or after, theimmunotoxin exposure step. The thymic apoptosis signal can be high dosecorticosteroids (also referred to as “immunosuppressants” in thiscontext). The thymic apoptosis signal can be lymphoid irradiation.

In a further example of the method of inducing tolerance, thymicinjection of donor leukocytes or lymphocytes having MHC antigen of thesame haplotype as the MHC of the donor cell can be administered to therecipient. Thymic injection of a saline solution or a crystalloid orcolloid solution to disrupt thymic integrity and increase access ofimmunotoxin to the thymus can also be beneficial.

The present tolerance induction method can also include administering animmunosuppressant compound before, at the same time, or after, theimmunotoxin exposure step. The immunosuppressant compound can bedeoxyspergualin, cyclosporin or other cyclophylins, mycophenolatemofetil (Roche), FK506, IL-12 inhibitors or other knownimmunosuppressants. The method of inducing immune tolerance can furthercomprise administering donor bone marrow at the same time, or after, theexposure step.

Any one, two, or more of these adjunct therapies can be used together inthe present tolerance induction method. Thus, the invention includes atleast six methods of inducing tolerance using immunotoxin (IT): (1)tolerance induction by administering IT alone; (2) tolerance inductionby administering IT plus other drugs that alter thymic function such ashigh dose corticosteroids; (3) tolerance induction by administering ITplus immunosuppressant drugs such as cyclosporin (4) tolerance inductionby administering IT plus other drugs that alter thymic function, plusimmunosuppressant drugs; (5) tolerance induction by administering IT andbone marrow; and (6) tolerance induction by administering IT plus bonemarrow, plus other drugs that alter thymic function, plusimmunosuppressant drugs. The adjunct therapy can be administered before,at the same time or after the administration of immunotoxin. Differentadjunct therapies can be administered to the recipient at differenttimes or at the same time in relation to the transplant event or theadministration of immunotoxin, as further described below.

Because the immunosuppressant can be administered before the immunotoxinand/or other treatments, the present method can be used with a patientthat has undergone an organ transplant and is on an immunosuppressantregimen. This presents a significant opportunity to reduce or eliminatetraditional immunosuppressant therapy and its well documented negativeside-effects. Also, as described below, treatment withimmunosuppressants prior to transplantation could be particularly usefulin cadaveric transplants. In such a setting of pre-transplant treatmentwith immunosuppressant, the administration of immunotoxin can beadvantageously delayed for up to seven or more dayspost-transplantation.

An example of a schedule of immunotoxin and immunosuppressantadministration for patients receiving live organ transplants is asfollows:

day -7 to day 0: begin immunosuppressant treatment;

day 0 : perform transplant;

day 7 : begin immunotoxin and non-toxic DT toxin treatment;

day 9 : end immunotoxin treatment;

day 11 : end immunosuppressant treatment.

In another example, non-toxic DT mutant is administered seven daysbefore the transplant and immunotoxin is administered seven days afterthe transplant.

The immunotoxin injection can also be made within a week or two prior tothe donor cell treatment. If the donor organ or cell from donor organ isfrom a live donor, the immunotoxin is preferably administered from 15hours to 7 days before the transplanting step. If the donor organ iskidney or kidney cells and is from a cadaver, the immunotoxin ispreferably administered from 6 to 15 hours before the transplantingstep. If the donor organ or cell from the donor organ is from a cadaverand is selected from the group consisting of heart, lung, liver,pancreas, pancreatic islets and intestine, the immunotoxin is preferablyadministered from 0 to 6 hours before the transplanting step. Forpractical reasons immunotoxin treatment and transplantation generallytake place at about the same time (e.g., within 15 hours), becauseadvanced planning for cadaveric transplants is difficult. Variousschedules of apoptotic and immunosuppressant therapies can be used withthe above methods. In any of the above scenarios, donor bone marrow, ifdesired, can be administered at approximately the time of the transplantor after.

The preferred doses of the immunotoxin are those sufficient to depleteperipheral blood T-cell levels to 80%, preferably 90% (or especiallypreferably 95% or higher) of preinjection levels. This should requiremg/kg levels for humans similar to those for monkeys (e.g. 0.15 mg/kg to0.2 mg/kg body weight), which toxicity studies indicate should be welltolerated by humans. Thus, the immunotoxin can be administered to safelyreduce the recipients T cell population.

Method of Treating Graft-Versus-Host Disease.

In another embodiment, the invention relates to a method of treating animmune system disorder not involving T cell proliferation which isamenable to T cell suppression. More specifically, a method of treatinggraft-versus-host disease in an animal is also provided. It comprisesadministering to the animal an immunotoxin comprising a diphtheria toxinbinding mutant moiety and an antibody moiety which routes by theanti-CD3 pathway, or derivatives thereof under conditions such that thegraft-versus-host disease is treated, i.e., the symptoms of thegraft-versus-host disease improve. Alternatively, as further described,a non-toxic DT mutant such as DTM2 or CRM197 (or mutants havingcombinations of the mutations in CRM9 and CRM197) can first beadministered followed by the immunotoxin. This method can use any of theimmunotoxins or non-toxic DT mutants described herein with the dosagesand modes of administration as described herein or otherwise determinedby the practitioner.

GVHD is a morbid complication of bone marrow transplantation which isoften performed as anti-leukemia/lymphoma therapy. GVHD is caused bycirculating donor T cells within the host which are acquired in bonemarrow grafts unless specifically depleted prior to grafting (Gale andButturini (1988) Bone Marrow Transplant 3:185; Devergie et al. (1990)ibid 5:379; Filipovich et al. (1987) Transplantation 44). Successfuldonor T cell depletion techniques have been associated with a higherfrequency of graft rejection and leukemia relapses (Gale and Butturini(1988) Bone Marrow Transplant 3:185; Devergie et al. (1990) ibid 5:379;Filipovich et al. (1987) Transplantation 44). Therefore, the donor Tcells appear to aid engraftment and to provide a graft-versus-leukemiaeffect as well as causing GVHD. Because the T cell burden following bonemarrow transplantation is low for the first 14 days (<10% of normal) thelog kill of donor T cells would be proportionally enhanced (Marsh andNeville (1987) Ann. N.Y. Acad. Sci. 507:165; Yan et al., submitted; Galeand Butturini (1988) Bone Marrow Transplant 3:185; Devergie et al.(1990) ibid 5:379; Filipovich et al. (1987) Transplantation 44). It isexpected that donor T cells can be eliminated at set times during theearly post transplantation period using the present method. In this waythe useful attributes of grafted T cells might be maximized and theharmful effects minimized.

Method of Treating an Autoimnune Disease.

Another embodiment of the invention provides a method of treating anautoimmune disease in an animal comprising administering to the animalan immunotoxin comprising a diphtheria toxin binding mutant moiety andan antibody moiety which routes by the anti-CD3 pathway, or derivativesthereof, under conditions such that the autoimmune disease is treated,e.g., the symptoms of the autoimmune disease improve. A further methodof treating an autoimmune disease in an animal comprises administeringto the animal a non-toxic mutant of diphtheria toxin followed by anantibody CRM9 conjugate which routes by the anti-CD3 pathway, orderivatives thereof, under conditions such that the autoimmune diseaseis treated. This method can use any of the immunotoxins or non-toxic DTmutants described herein with the dosages and modes of administration asdescribed herein or otherwise determined by the practitioner.

Method of Treating T Cell Leukemias or Lymphomas.

A further embodiment of the invention provides a method of treating Tcell leukemias or lymphomas which carry the CD3 epitope in an animalcomprising administering to the animal an immunotoxin comprising abinding site mutant of diphtheria toxin moiety and an antibody moietywhich routes by the anti-CD3 pathway, or derivatives thereof, underconditions such that the T cell leukemias or lymphomas are treated.Alternatively, a further embodiment is a method of treating T cellleukemias or lymphomas in an animal comprising administering to theanimal a non-toxic mutant of diphtheria toxin followed by anantibody-CRM9 conjugate which routes by the anti-CD3 pathway, orderivatives thereof, under conditions such that the T cell leukemias orlymphomas are treated. This method can use any of the immunotoxins ornon-toxic DT mutants described herein with the dosages and modes ofadministration as described herein or otherwise determined by thepractitioner.

Method of Treating Acquired Immunodeficiency Syndrome.

A method is provided for treating acquired immunodeficiency syndrome inan animal, comprising administering to the animal an immunotoxincomprising a diphtheria toxin binding mutant moiety and an antibodymoiety which routes by the anti-CD3 pathway, or derivatives thereofunder conditions such that the acquired immunodeficiency syndrome istreated. Alternatively, a method of treating acquired immunodeficiencysyndrome in an animal, comprising administering to the animal anon-toxic mutant of diphtheria toxin followed by an antibody-CRM9conjugate which routes by the anti-CD3 pathway or derivatives thereofunder conditions such that the acquired immunodeficiency syndrome istreated is provided. This method can use any of the immunotoxins ornon-toxic DT mutants described herein with the dosages and modes ofadministration as described herein or otherwise determined by thepractitioner. However, anti-Vβ₁₂ is a likely conjugate for use in thismethod.

Radiation induced T cell ablation with concomitant high dose zidovudinetherapy followed by bone marrow transplantation has been reported toeradicate HIV-1 infection in one case (Holland et al. (1989) Ann. Int.Med. 111:973). Cyclophosphamide, a T cell suppressive reagent, has beenshown to be beneficial in treating murine AIDS (Simard and Joliceur(1991) Science 251:305). Anti-CD3-CRM9 provides extensive T cellablation without the requirement of bone marrow reconstitution.

In any of the methods recited, a H1 histamine blocking agent such asBenadryl or Tagevil can be administered I.V. prior to administering thenon-toxic mutant to minimize any possibility of an anaphylacticreaction. No evidence of anaphylactic reaction was noted in the primateexperiments described in the Examples. However, the H1 histamine blockercan be administered as a precaution with no significant disadvantage.

The immunotoxin described here is more toxic on a weight basis thanhemi-immunotoxins, but at tolerated doses exhibits an apparent log killof targeted cells at target cell burdens encountered clinically. Thisconstitutes a favorable therapeutic margin. Most human sera containanti-DT neutralizing antibodies from childhood immunization (Johnson etal. (1989) J. Neurosurg. 70:240). To compensate for this the therapeuticdose of anti-CD3-CRM9 can be appropriately raised without affecting thetherapeutic margin. The doses for immunotoxin and, where used, thenon-toxic DT mutant are described in the Examples.

The present invention will be illustrated in further detail in thefollowing non-limiting examples.

EXAMPLE 1 Establishment of Tumors

The experimental design of the studies that give rise to the presentinvention was dictated by the goal of having an animal model as closelyrelevant to human in vivo tumor therapy as possible. In order tominimize the host killer cell immune response, bg/nu/xid strain of nudemice were used (Kamel-Reid and Dick (1988) Science 242:1706). The humanT cell leukemia cell line, Jurkat, was chosen because of previousstudies with this line and its relatively normal average complement ofCD3 receptors (Preijers et al. (1988) Scand. J. Immunol. 27:553). Theline was not cloned so that receptor variation among individual cellsexisted (FIG. 1 legend). A scheme was developed whereby well establishedtumors of constant mass equal to 0.1% of body weight (=4×10⁷ cells)could be achieved 7 days after inoculation of Jurkat cells (see FIG. 1and Dillman et al. (1988) Cancer Res. 15:5632). This required priorirradiation and inoculation with lethally irradiated helper feeder cells(see FIG. 1 and Dillman et al. (1988) Cancer Res. 15:5632).

EXAMPLE 2 Guinea Pig Studies

Immunotoxin toxicity studies were performed in guinea pigs, an animal(like humans) with a high sensitivity to diphtheria toxin (mice arehighly resistant to diphtheria toxin). Therapy of CRM9 conjugates wasset at ½ the guinea pig minimum lethal dose. In this study, minimumlethal dose (MLD) is defined as the minimum tested dose which results inboth non-survivors and survivors over a 4 week evaluation period. Allanimals survive when a MLD is reduced by 0.5. MLD was evaluated inguinea pigs (300-1000 g) by subcutaneous injection. The following MLDswere found and are listed as μg of toxin/kg body weight; DT, 0.15; CRM9,30; anti-CD5-DT (cleavable), 0.65; anti-CD5-CRM9 (non-cleavable), 150.Finally, the therapeutic efficacy of the immunotoxin treatment inproducing tumor regressions was compared to graded doses of whole bodyirradiation which resulted in similar tumor regressions.

EXAMPLE 3 Comparison of Immunotoxins

Several types of immunotoxins were compared in this study. They weresynthesized as previously described by thiolating both the monoclonalantibody moiety and the toxin moiety and then crosslinking thebismaleimide crosslinkers (Neville et al. (1989) J. Biol. Chem.264:14653). Purification was performed by size exclusion HPLC columnsand fractions containing 1:1 toxin:antibody mol ratios were isolated forthese studies. Conjugates made with an acid-labile crosslinkerbismaleimidoethoxy propane were compared with a non-cleavable,bismaleimidohexane. Conjugates made with this cleavable crosslinker havebeen shown to hydrolyze within the acidifying endosome releasing freetoxin moieties with half-times of hydrolysis measured at pH 5.5 of 36min (Neville et al. (1989) J. Biol. Chem. 264:14653).

The results of this study are tabulated in Table I. Non-treatment groupssuch as group 10, groups treated with anti-CD5 immunotoxins (groups 5and 6), and group 4 treated with a mixture of anti-CD3 and CRM9 did notshow regression. The vascularized tumor nodules that weighed 20 mg onday 7 grew to between 1.5 to 7.8 g on day 37 and weighed between 7.9and. 11.6 on day 56. No late spontaneous regressions were noted. Incontrast, group 1 consisting of treatment with anti-CD3-CRM9non-cleavable conjugate (NC) given at 25 μg/kg on days 7, 8, and 9 (seeFIG. 1 time line) showed only 1 tumor out of 6 by day 37. Some of theremaining animals were subject to autopsy and they failed to revealresidual tumor or even scaring. Tumors identified as regressed on day 37by superficial inspection did not reappear during the course of thestudy (56 days).

TABLE 1 IMMUNOTOXIN AND RADIATION TREATMENT ON SUBCUTANEOUS HUMAN T CELLTUMORS (JURKAT) IN NUDE MICE Dose Animals Bearing Tumors % Tumor GroupTreatment (intraperitoneal) At Day 37/Group Animals Regressions  1Anti-CD3 − CRM9 (NC)^(a) 25 μg/kg. × 3d 1/6 83  2 Anti-CD3 − CRM9 (NC)19 μg/kg. × 2d 1/4 75 Anti-CD5 − CRM9 (C) 19 μg/kg. × 2d  3 Anti-CD3 −CRM9 (C) 25 μg/kg. × 3d 2/4 50  4 Anti-CD3 + CRM9 25 μg/kg. × 3d 4/4 0 5 Anti-CD5 − CRM9 (C) 25 μg/kg. × 3d 5/5 0  6 Anti-CD5 − DT (NC) 25μg/kg. × 1d 9/9 0  7 γ radiation ¹³⁷Cs 400 cGy 2/2 0  8 γ radiation¹³⁷Cs 500 cGy 3/6 50  9 γ radiation ¹³⁷Cs 600 cGy  0/2^(b) 100 10 None6/6 0 ^(a)Anti-CD3 refers to the monoclonal antibody UCHT1 and waspurchased from Oxoid USA, Inc. Anti-CDS refers to the monoclonalantibody T101 and was a gift from Hybritech (San Diego). NC and C refer,respectively, to non-cleavable and cleavable conjugates. ^(b)Theseanimals were evaluated on days 10 and 13 at the time of death fromradiation sickness.

The cleavable crosslinker confers no therapeutic advantage toanti-CD3-CRM9 immunotoxins and may be less effective (group 3).Cleavable crosslinkers confer some advantage with anti-CD5-CRM9conjugate in vitro (5) but had no effect in this in vivo system (group5), and lacked significant potentiating effect when administered withanti-CD3-CRM9 (group 2). The cleavable crosslinker conferred a markedtherapeutic advantage to anti-CD5 wild type toxin conjugates and tumorregressions were achieved. However, in these cases the guinea pig toxicdose was exceeded. A single dose on day 7 of cleavable anti-CD5-DT at 6μg/kg produced 8/10 tumor regressions while a cleavable conjugate madewith an irrelevant antibody (OX8) produced no regressions (4/4).However, this dose exceeded the guinea pig MLD by 9 fold. A rescuestrategy was tried in which the above conjugate dose was givenintravenously followed by DT antitoxin 4 hours later (alsointravenously). The 4 hr rescue could not raise the MLD above 0.65μg/kg. The 1 hr rescue could not raise the MLD above 0.65 μg/kg. The 1hr rescue raised the MLD to 36 μg/kg, however, there were no tumorregressions in 10 mice receiving 21.5 μg/kg of the cleavable anti-CD5-DTconjugate.

In groups 7-9 increasing single doses of whole body radiation (102cGy/min) were given to animals bearing 3×3×5 mm tumors. At 400 cGy nocomplete regressions occurred. At 500 cGy 50% complete tumor regressionsoccurred. At 600 cGy 100% regression was achieved as judged on day 10and 13 when the animals died from radiation sickness. (Groups 7-9 didnot receive prior radiation and tumor takes were less than 100%). Itappears that the 75 μg/kg anti-CD3-CRM9 (NC) immunotoxin is equal intherapeutic power to between 500 and 600 cGy of radiation.

EXAMPLE 4 Estimation of Cell Kill

The actual cell kill achieved by the radiation and the immunotoxin canbe estimated by assuming radiation single hit inactivation kineticsalong with a D₃₇ value for the radiation. A value for D₃₇ of 70-80 cGywith n=1.2-3 is not unreasonable for a rapidly dividing helper T cell.D₃₇ is the dose of radiation which reduces the fraction of survivingcells to 1/e as extrapolated from the linear portion of the logsurvivors vs. dose curve and n is the intercept at 0 dose (Anderson andWarner (1976) in Adv. Immunol., Academic Press Inc., 24:257). At a doseof 550 cGy the fraction of surviving cells is calculated to be about10³. Since a majority of tumors completely regress at this dose, it isestimated that both therapies are producing an approximate 3 log kill.(The remaining cells, 4×10⁷×10³=4×10⁴ cells apparently cannot maintainthe tumor, i.e., the in vivo plating efficiency is low, a fairly typicalsituation in the nude mouse xenograft system.) The reliability of this 3log kill estimate has been verified by determining the tissue cultureplating efficiency by limiting dilution of 7 day established Jurkattumors (following dispersal) and tumors exposed 18 hours earlier in vivoto 600 cGy. Plating efficiencies were 0.14 and 1.4×10⁴, respectively.(Plating efficiency is the reciprocal of the minimum average number ofcells per well which will grow to form one colony.

It should be emphasized that with high affinity holo-immunotoxins thecell kill is inversely proportional to the target cell number. Thispresumably occurs because receptors are undersaturated at tolerateddoses and free conjugate concentration falls with increasing target cellburden (Marsh and Neville (1987) Ann. N.Y. Acad. Sci. 507:165; Yan etal. (1991) Bioconjugate Chem. 2:207). To put this in perspective, thetumor burden in this study is almost equal to the number of T cells in amouse (=10⁸). It can be expected that a tolerated dose of anti-CD3-CRM9immunotoxin can achieve an in vivo 3 log depletion of a normal number ofCD3 positive T cells.

EXAMPLE 5 Call Depletion in Rhesus Monkeys Induced by FN18-CRM9

FN18-CRM9 conjugate.

Conjugation of anti-Vβ and anti-Vα IgG monoclonal antibodies to CRM9 isperformed by the same methods used to conjugate anti-CD3 to CRM9 using anon-cleavable linker such as bismaleimidohexane and previously describedin detail (Neville et al. (1988) J. Biol. Chem. 264:14653-61). Themonoclonal antibody FN18 is the monkey equivalent of the human anti-CD3(UCHT1) and is known to bind the same CD3 receptor epitopes (ε and γ) asbound by the human CD3 antibody and is the same isotype as the human CD3antibody. Thus, in terms of the parameters relevant for predictingsuccessful T cell depletion, the present CD3-CRM9 conjugate andFN18-CRM9 are expected to have the same activity.

Administration.

Conjugates can be administered as an I.V. bolus in a carrier consistingof 0. 1M Na₂SO₄+0.01M phosphate buffer, pH 7.4 plus 1 part in 50 ofserum previously obtained from the subject. The dose schedule is everyother or third day for 3 to 6 days. The total dose is preferably from 25to 200 micrograms of toxin per kg of body weight.

The actual dose of FN18-CRM9 used was equal to 0.167 of the minimumlethal dose (MLD) in guinea pigs. Since the estimation of the MLD wasperformed in an animal lacking an immunotoxin target cell population(guinea pigs), the true MLD of FN18-CRM9 and anti-CD3-CRM9 is expectedto be higher in monkeys and humans than in guinea pigs.

T Cell Kill.

Helper T cell (CD4+cells) numbers in peripheral blood fell dramaticallyafter the initial administration of FN18-CRM9 in two rhesus monkeys. Tcell counts began to rise by day 4 (sampled just prior to the seconddose of FN18-CRM9). On day 5 in monkey 8629, CD4+cells were depressedbelow the limit of detection (<50 cells/mm³). Cells remained below orequal to 200/mm³ out to day 21. This low level of CD4+ cells isassociated with profound immunodeficiency in humans and in monkeys(Nooij and Jonker (1987) Eur. J. Immunol. 17:1089-1093). The remarkablefeature of this study is the long duration of helper T cell depletion(day 21) with respect to the last administration of immunotoxin (day 4)since intravenously administered immunotoxins were cleared from thevascular system with half-lives <9 hours (Rostain-Capaillon and Casellas(1990) Cancer Research 50:2909-2916), the effect outlasting circulatingimmunotoxin. This is in contrast to T cell depletion induced byunconjugated anti-CD3 antibodies (Nooij and Jonker (1987) Eur. J.Immunol. 17:1089-1093).

In monkey 1WS the second dose of conjugate only appeared to result in adiminished rate of CD4+ cell recovery. However, CD4+ cells were stillfewer than normal at day 21. The blunted response of monkey 1WS to thesecond dose of immunotoxin was found to be due to a preexistingimmunization of this animal to the toxin. Monkey 1WS had a significantpre-treatment anti-diphtheria toxin titer as revealed by a Western blotassay. This titer was markedly increased at day 5, indicative of aclassic secondary response. In contrast, monkey 8629 had no detectablepre-treatment titer and only a trace titer by day 5 and a moderate titerby day 28.

The specificity of FN18-CRM9 toward T cells can be seen by comparing thetotal white blood cell (WBC) count in the same two monkeys. WBCs fell,but only to 45% of baseline value on day 2 compared to 6% of baselinevalues for the CD4+ T cell subset. Most of the fall in WBC values can beaccounted for by the T cell component of the WBC population (≈40%).However, B cells are initially depleted after FN18-CRM9 although thesecells recover more quickly. FN18 is an IgG, isotype and as such is knownto bind to Fc_(II) receptors present on B cells and macrophages with lowaffinity. The FN18-CRM9 depletion of B cells indicates that significantinteractions between the Fc portion of the FN18 antibody and B cells istaking place.

The peripheral T cell depletion induced by unconjugated FN18 at a doseknown to produce immunosuppression 0.2 mg/kg/day (Nooij and Jonker(1987) Eur. J. Immunol. 17:1089-1093) was compared to the immunotoxinFN18-CRM9 administered at 1/9th the FN18 dose. Peripheral CD4+T celldepletion is more pronounced and more long-lasting with the conjugate.The demonstration that FN18-CRM9 reduces peripheral helper T cell subset(CD4+) to levels less than or equal to 200 cell/mm³ for a period as longas 21 days demonstrates that this immunotoxin and its anti-human analogsare effective immunosuppressive reagents.

The demonstration that FN18-CRM9 is a potent agent for inducing T celldepletion in non-human primates demonstrates that an anti-human homologof FN18-CRM9, UCHT1-CRM9 (Oxoid USA, Charlotte, N.C.) for example, is apotent agent for inducing T cell depletion in humans.

The Fc binding region of anti-TCR/CD3 monoclonals may or may not beneeded to induce T cell depletion when the anti-TCR/CD3 monoclonals areconjugated to CRM9. The Fc_(II) binding regions can be removed, forexample, by forming the conjugates with F(ab′)₂ derivatives as isindicated in the literature (Thorpe et al. (1985) J. Nat'l. Cancer Inst.75:151-159). In addition, anti-TCR/CD3 IgA switch variants such asmonoclonal antibody T3. A may be used (Ponticelli et al. (1990)Transplantation 50:889-892). These avoid rapid vascular clearancecharacteristic of F(ab′)2 immunotoxins. F(ab′)₂ and IgA switch variantsof anti-TCR/CD3-CRM9 immunotoxins are therefore derivative anti-TCR/CD3immunotoxins. These derivatives will avoid the B cell interaction notedand can increase specificity. However, IgG_(2a) switch variants willmaximize T cell activation through the Fc_(I), receptor and may beuseful in certain situations where T cell activation aids immunotoxininduced toxicity.

General methods to make antibodies lacking the Fc region or to makeantibodies which are humanized are set forth in Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor,. N.Y., 1988. Thus, as used in the claims, antibody canmean the entire antibody or any portion of the antibody sufficient forspecific antigen or receptor binding.

EXAMPLE 6 Treatment of Autoimmune Diseases Using Other Antibody-CRM9Conjugates which Route by the Anti-CD3 Pathway

Since receptor recycling is a requirement for effective CRM9 basedimmunotoxins and since TCR/CD3 recycles as a unit, antibodies directedat other epitopes on TCR/CD3 will constitute effective derivatives, inparticular antibodies directed at the approximately 50 Vβ subsetfamilies or the approximately equal number Vα subsets can be used toconjugate CRM9 and ablate specific Vβ or Vα subsets in vivo. Inaddition, in some cases it will be desirable to develop specificmonoclonal antibodies reacting with unique rearrangements of either theVα or Vβ subset families.

The advantage of targeting the specific Vβ or Vα subset(s) as opposed tothe entire T cell population is twofold: (1) Elimination of a Vβ subsetdoes not create a generalized immunodeficiency, only a hole in theimmune repertoire is generated. Therefore, the ability to ward off mostinfections and maintain immune surveillance of most malignanttransformations would remain intact. (2) Immunotoxin log kill increaseslinearly as the target cell burden decreases, assuming dose isunchanged. A 50-fold increase in log kill can be obtained as the targetis changed from the entire set of T cells to a single Vβ subset.However, due to (1) the high affinity of binding of these immunotoxins,(2) the very low total dose given which is below target cell receptorsaturation and (3) the irreversible nature of the endocytotic process,the target cells deplete the effective dose and this depletion decreasesas target burden decreases. Since the log kill is exponential ineffective dose, much higher increases in log kill than 50-fold onchanging the target from T cells to a Vβ subset can occur. The expectedincrease in log kill will only occur if the immunotoxin is specific forthe defined target. Extraneous interactions with other cell types viathe antibody Fc piece is preferably eliminated.

Because HIV has been shown to preferentially infect one (Vβ₁₂) or a fewof the 20 Vβ subset families providing a small T cell reservoir of HIVreplication, and because HIV infection apparently involves an unknownsuperantigen, CRM9 based immunotoxins directed at these specific Vβsubsets such as anti-Vβ₁₂-CRM⁹ can reduce the HIV virus load. Inaddition, total ablation of a Vβ subset in the presence of an endogenoussuperantigen can lead to long term ablation of the subset since maturingT cells are negatively selected in the presence of endogenoussuperantigens. Since the specific Vβ subset responding to thesuperantigen is eliminated, infection cannot take place.

The two strategies that can be utilized for using anti-Vβ₁₂-CRM9immunotoxins to treat HIV infections are (1) treatment depleting thesusceptible Vβ subset to an extent where continued infection cannot bemaintained and (2) treatment to the extent that all or nearly all of theVβ₁₂ subset is eradicated.

Anti-human Vβ monoclonal antibodies such as S5-11 (anti-Vβ₁₂) areavailable (T Cell Sciences, Cambridge, Mass.) and can be conjugated toCRM9 by standard methodologies.

Briefly, as in Example 5, conjugation of anti-Vβ and anti-Vα IgGmonoclonal antibodies to CRM9 is performed by the same methods used toconjugate anti-CD3 to CRM9 using a non-cleavable linker such asbismaleimidohexase and previously described in detail (Neville et al.(1988) J. of Biol. Chem. 264:14653-61).

Conjugates can be administered as an I.V. bolus in a carrier consistingof 0.1M Na₂SO₄+0.01M phosphate buffer, pH 7.4 plus 1 part in 50 of serumpreviously obtained from the patient. The dose schedule is every otheror third day for 3 to 6 days. The total dose is preferably from 25 to200 micrograms of toxin per kg of body weight, but may be increased ifanti-diphtheria toxin antibodies are present in the patient's sera insignificant amounts.

Other Vβ or Vα subsets which may be found to be associated with HIVinfection can be treated in the same manner described herein byconjugating the CRM9 to the antibody specifically reactive with theappropriate Vβ or Vα subset.

EXAMPLE 7 T Cell Depletion and Immunosuppression in Monkeys Using theImmunotoxin Anti-CD3-CRM9.

CRM9 is a diphtheria toxin (DT) binding site mutant and forms the basisof the anti-T cell immunotoxin anti-CD3-CRM9. This immunotoxin has beenconstructed against human and rhesus T cells and has shown above to kill3 logs of human T cells in a nude mouse xenograft system. The presentexample demonstrates a 2 log kill of T cells in rhesus monkey lymphnodes that is also shown to produce prolongation of skin allograftrejection in monkeys.

Humans are immunized against diphtheria toxin by exposure to DPTvaccines in childhood. This long lasting immunity may interfere with theefficacy of DT based immunotoxins. Many monkeys are immunized against DTby natural exposure to toxin producing Corynebacterium. The presentmethod addresses any potential interference of pre-existing DTantibodies with the activity of the present immunotoxins.

ELISA

ELISA assays were performed in order to determine the levels of anti-DTtiters existing in 9 individuals in a population ages 27 to 55. Therewere 3 individuals with titers of 1:100 (low) and 6 with titers of1:1000 (moderate).

Rhesus monkeys were screened by the same assay and a 1:1000 titeredmonkey was selected.

Administration of Non-Toxic Diphtheria Toxin Mutant.

Monkeys were treated by I.V. route 5 min prior to the immunotoxin dosewith a 100 fold excess of CRM197 over the CRM9 content of theimmunotoxin to be administered. Just prior to administering CRM197, a Hihistamine blocking agent such as Benadryl or Tagevil was given I.V. tominimize any possibility of an anaphylactic reaction (for Benadryl 4mg/kg). No histaminic reaction was detected.

Anti-CD3-CRM9 was given at a total dose between 0.1 and 0.2 mg/kg (toxinweight) in 3 equally divided doses (approximately 0.033 mg/kg) on 3consecutive days. In these monkeys, the total dose of immunotoxin was0.1 mg/kg.

Table 1 shows a comparison of the efficacy of anti-CD3-CRM9 in monkeysby comparing the decrease in the lymph node T/B cell ratio (a measure oflymph node T cell depletion) and the immunosuppressive effect of theimmunotoxin as judged by prolongation of mismatched skin graft survival.Effects on the survival of skin grafts is a clear indicator of thegeneral effect a given treatment has on the subject's immune system.

The monkey with the preexisting anti-DT titer that was pretreated withCRM197 shows the same level of T/B cell inversion as in the negativetitered monkey. Skin graft survival was significantly prolonged over thetitered monkey treated without CRM197. The failure to achieve aprolongation of graft survival equal to the negatively titered monkey islikely due to the lower weight of this monkey which causes T cells torepopulate faster, in this case 3-4 days faster, due to the largerthymic T cell precursor pool in younger animals. Age related effectssuch as these can be compensated for by modification of dosage levelsand timing of administration.

TABLE 2 Efficacy of Anti-CD3 − CRM9 With and Without CRM197 In RhesusMonkeys With Positive and Negative Anti-Diphtheria Toxin Titers. PostTreatment* Lymph node T/B Day(s) of Skin Monkey Weight kg Anti-DT TiterTreatment Cell Ratio Graft Survival historical 4-7 N/A None 2.1-2.4⁺ 9.5± 08^($) controls B65 5.1 neg anti-CD3 1.8 12, 12 8838 5.1 neg anti-CD3− CRM9 0.14^(xx) 19, 20 M93 5.1 1:1000 anti-CD3 − CRM9 0.57 11, 12 C811.0 1:1000 CRM197 + anti- 0.20 14, 15 CD3 − CRM9 *All monkeys receivedthe same dose of immunotoxin 0.1 mg/kg total in divided doses on day 0,1 and 2. Lymph node sampled on day 3. CRM197 when given in 100 foldexcess over CRM9 content. ⁺In this study untreated animals show thislymph node T/B ratio ^($)Historical controls at TNO, Rijswijk^(xx)Anti-CD3 given at the same mol. dose as anti-CD3 − CRM9

EXAMPLE 8 Immunotoxin UCHT1-CRM9 for the Treatment of Steroid ResistantGraft-Versus-Host Disease

Treatment protocols for this type of disease can be expected to last ayear, with Patients being followed for at least 5 years.

Characterization of UCHT1-CRM9 and CRM197.

UCHT1-CRM9 is a covalent 1:1 conjugate of anti-human CD3 IgG1 monoclonalantibody and CRM9. The conjugate is synthesized, purified, sterilefiltered and assayed for concentration, biological efficacy towardtarget cells and non-target cell toxicity by standardized cultureassays. The method of synthesis, purification assay are identical tothat used for FN18-CRM9 which was used in the pre-clinical monkeystudies described in Examples 5-7.

CRM9 and CRM197 are produced by the Biotechnology Unit, NIH and purifiedby the Cooperating Facility. UCHT1 is produced in mouse ascites fluidand is purified by affinity chromatography over Protein A Sepharose. Thesynthesis, purification and storage of UCHT1-CRM9 is performed in adedicated secure area. UCHT1-CRM9 is purified in 2 mg lots which arepooled and stored at 4° C. Shelf life is documented to be five months atfull biological potency but does not exceed 4 months for this study.Preferably, most of the immunotoxin is used within 3 months ofsynthesis.

Patient Population.

The patient population consists of individuals suffering from steroidresistant GVHD whose prognosis is poor. Patients are assayed foranti-CRM9 (anti-DT) titers and antibodies to murine immunoglobulin.Patients having anti-CRM9 titers of 1:1000 and below are treatedaccording to the present protocol. Patients who have a history ofreceiving murine immunoglobulins or who exhibit positive anti-Ig titersmay require special consideration.

Dosage of CRM9 Immunotoxin and Non-Toxic Mutant.

UCHT1-CRM9 is administered at a dose which is 1/10 or less of theestimated minimum lethal dose (MLD) in a T lymphopenic patient. The MLDis expected to be at least 0.15 mg/kg (CRM9 content) based on the MLD of0.15 mg/kg of IgG1-CRM9 in guinea pigs which lack a target cellpopulation for the IgG1. (The presence of target cells in humans raisesthe MLD by providing a sink for the immunotoxin.) The optimal doseschedule has been found in monkeys to be administration on 3 consecutivedays in 3 equally divided doses, and this schedule can be usedthroughout the treatment period. This permits administration of thetotal dose before any rise in pre-existing antitoxin titers due to asecondary response. In addition, the initial repopulation from thethymus is also eliminated, thus, further lowering the total T lymphocytepool. Therefore, a total of 0.0125 mg/kg in three equally divided dosesis given to the patient. This dose does induces T cell depletion inmonkeys so that monitoring of T cell subsets and signs and symptoms ofGVHD is relevant at the lowest dose. For the administration of this dosepatients with anti-CRM9 titers of 1:100 or less will be treated. Thispermits pretreatment doses of CRM197 at 0.33 mg/kg or 1/10 the doseeasily tolerated in monkeys. A second dosage group can include patientsselected for antitoxin titers of 1:330 or less to whom CRM197 will begiven at 1.0 mg/kg. A third dosage group can include patients with1:1000 antitoxin titers or less will be given CRM197 at 3.3 mg/kg, adose expected to be tolerable in humans, because it is easily toleratedby monkeys (see Example 7). The monkey MLD data should be very similarto humans on a per weight basis. However, GVHD patients are expected tobe more like guinea pigs, because they have a smaller target cellpopulation compared to non-GVHD patients.

Dose escalation can be tested by increasing the dose by a factor of 1.5.The following table exemplifies such a dose escalation test. For examplethree patients are used in each dosage group. There is a 3 to 4 weekdelay between each patient so that any late toxicity is detected beforea dosage group is completed:

CRM9 Dose each day Total Dose Patient # mg/kg mg/kg Week ending 1, 2, 30.00417 0.0125 12 4, 5, 6 0.00636 0.019 24 7, 8, 9 0.0083 0.028 36 10,11, 12 0.0125 0.042 48

Assuming each patient weighs on the average 70 kg, the first dosagegroup will consume 2.6 mg of the CRM9 immunotoxin, and will be suppliedas a pool of two 2 mg batches. The second group will consume 3.9 mg andwill also be supplied as 2 pooled batches. The third group will require5.9 mg and will be supplied as three pooled batches. The fourth groupwill require 8.9 mg and will be supplied as three pooled batches and anadditional two pooled batches.

Administration.

Prior to administering CRM197 a Hi histamine blocking agent such asBenadryl or Tagevil is given I.V. to minimize any possibility of ananaphylactic reaction (for Benadryl 4 mg/kg). The CRM197 is administeredI.V. in a 5 mg/ml sterile filtered solution in phosphate buffered salinepH 7.4 (PBS) over a 5 min time period. The immunotoxin is then givenI.V. at 0.2 mg/ml over 2 min time period in a sterile filtered solutionof 0.90 mM sodium sulfate and 10 mM sodium phosphate pH 7.4.

Measurements of Biological Parameters.

The following parameters can be measured at various intervals duringtreatment (as exemplified by the schedule below):

-   A Cytokines, TNF alpha, gamma IFN, IL-6-   B Routine clinical chemistries-   C WBC, Hct, diff; lymphocyte subsets CD3, CD4, CD8, CD2, CD16, CD20-   D Body Weight-   E Immune function assays. ELISA assays of serum to    monitor antibody responses to UCHT1 (primary response) and CRM9    (secondary response). ELISA assays to monitor antibody responses to    polio and DPT reimmunizations done at 1 year following bone marrow    transplantation.

(before IT) Day 0 A, B, C, D, E Also A 2 hrs post Day 1 A, C, D Day 2 A,C, D Day 3 A, B, C, D Day 4 C, D Day 7 A, C, D Day 10 B, C Day 14 A, C,D Day 21 C, D Day 28 A, B, C, D, E Day 45 C, D Day 60, B, C, D, E

EXAMPLE 9 An anti-CD3 single-chain immunotoxin with a truncateddiphtheria toxin avoids inhibition by pre-existing antibodies in humanblood

The present Example examines the effect of human serum with pre-existinganti-DT antibodies on the toxicity of UCHT1-CRM9, an immunotoxindirected against CD3 molecules on T-lymphocytes. Sera with detectableanti-DT antibodies at 1:100 or greater dilutions inhibited theimmunotoxin toxicity. Experiments with radiolabeled-UCHT1-CRM9 indicatethat anti-DT antibodies partially block its binding to the cell surfaceas well as inhibit the translocation from the endosome to the cytosol.The inhibitory effect could be adsorbed using a full-length DT mutant orB-subfragment. A C-terminal truncation mutant could not adsorb theinhibitory effect, suggesting that the last 150 amino acids contain theepitope(s) recognized by the inhibitory antibodies.

Therefore, an anti-CD3 single-chain immunotoxin, sFv-DT390, was madewith a truncated DT. The IC₅₀ of sFv-DT390 was 4.8×10⁻¹¹ M, 1/16 thepotency of the divalent UCHT1-CRM9. More importantly, sFv-DT390 toxicitywas only slightly affected by the anti-DT antibodies in human sera.“sFv” and “scUCHT” both are singe chain antibodies containing thevariable region.

Mutated full-length and truncated diphtheria toxin (DT) molecules areused for making immunotoxins. These immunotoxins show strong cytotoxiceffects to their target cells, and some of them have already been usedin clinical trials (1-7).] Previously, an immunotoxin directed againstthe CD3ε molecule of the T-cell receptor complex, a pan T-cell markerwas constructed. This construct is made with a monoclonal antibody ofmouse-origin, UCHT1, and a binding site mutant of diphtheria toxin (DT),CRM9 (8). The immunotoxin, UCHT1-CRM9, is capable of regressingestablished xenografted human T-cell (Jurkat) tumors in nude mice (9). Arhesus monkey analog of UCHT1-CRM9, FN18-CRM9 was capable of not onlydepleting circulating T-cells but also depleting resident T-cells in thelymph nodes. This immunotoxin also delayed skin allograft rejection ascompared to antibody treatment and non-treatment controls.

In contrast with ricin and Pseudomonas exotoxin (PE) based immunotoxins,there is a potential problem using UCHT1-CRM9, or other DT-basedimmunotoxins, in the treatment of human diseases. Most people have beenimmunized against DT. Therefore these people have a pre-existing anti-DTantibody titer which could potentially inhibit or alter the efficacy ofthese immunotoxins. This limitation also occurred in rhesus monkeystudies. FN18-CRM9 could deplete T cells in the blood, but to a muchlesser extent in animals with anti-DT antibodies, and the T cellsrepopulated several days earlier compared to those monkeys withoutanti-DT titers. In order to overcome this antibody mediated inhibition,the first examination of the effect and the mechanism of human seracontaining anti-DT antibodies on UCHT1-CRM9 toxicity was done.

A DT point-mutant, a truncation mutant and DT-subfragments were used inan attempt to neutralize the anti-DT effect in human sera. Based on theneutralization data, a single-chain immunotoxin was constructed with aC-terminal deletion mutant of DT which is expected to bypass theinhibitory effect of the pre-existing anti-DT antibodies.

Cells.

Jurkat cells (ATCC) were maintained in RPMI 1640 supplemented with 10%fetal calf serum, 25 mM sodium bicarbonate and 50 μg/ml of gentamycinsulfate.

Serum and adsorbing molecules.

Goat anti-DT serum was provided by Dr Randall K. Holmes (USUHS,Bethesda, Md.). Human serum samples were provided by Dr. Henry McFarland(NINDS, NIH, Bethesda Md.). CRM197, an A-subfragment mutant (Gly 52 toGlu) of DT (see FIG. 2A), with no enzymatic activity (10) is availablefrom Biocine-IRIS (Siena, Italy). MSPΔ5, a truncation mutant (amino acid385) of DT with an additional 5 amino acids at the C-terminus wasprovided by Dr. Richard Youle (NINDS, NIH, Bethesda Md.). Purificationof the DT B-subfragment has been described (11). Immunotoxins-UCHT1-CRM9synthesis has been described (12).

The recombinant immunotoxin, sFv-DT390, was generated in two phases.First the coding sequences for the variable light (V_(L)) and variableheavy (V_(H)) chain regions of the UCHT1 antibody were amplified by atwo step protocol of RT-PCR using primers based on the publishedsequence (13). The 5′ V_(L) primer added a unique NcoI restrictionenzyme site while the 3′ V_(H) primer added a termination codon at the Jto constant region junction and an EcoRI site. The V_(L) region wasjoined to the V_(H) region by single-stranded overlap extension and thetwo regions are separated by a (Gly₃Ser)₄ linker that should allow forproper folding of the individual variable domains to form a functionantibody binding site (14). Second, genomic DNA was isolated from astrain of C. diphtheriae producing the DT mutant CRM9(C7[β^(htox-201tox-9h′)]) as described (15). This DNA was used for PCR.The 5′ primer was specific for the toxin gene beginning at the signalsequence and added a unique NdeI restriction site. The 3′ primer wasspecific for the DT sequence terminating at amino acid 390 and added anNcoI site in frame with the coding sequence. The PCR products weredigested with the appropriate restriction enzymes and cloned into the E.coli expression plasmid pET-17b (Novagen, Inc., Madison, Wis., USA)which had been linearized with NdeI and EcoRI. The resulting plasmid wasused to transformed E. coli BL21/DE3 cells. Cells were grown to an OD₅₉₀of 0.5, induced with 0.5 M IPTG (Invitrogen, San Diego, Calif., USA) andincubated for an additional 3 hours. The sFv-DT390 protein was isolatedin the soluble fraction after cells were broken with a French Press andthe lysate subjected to centrifugation at 35,000 X g.

Protein synthesis inhibition assay.

Inhibition assays were performed as described (12) with the followingmodifications. Immunotoxins were incubated for 30 minutes with theindicated serum sample or leucine free medium at room temperature priorto addition to cells. In some experiments the serum was pre-incubatedfor 30 minutes with an adsorbing molecule at the given concentrations tobind the antibodies. The immunotoxin/serum mixture was incubated withJurkat cells (5×10⁴ cells/well in 96 well plate) for 20 hours. A 1 hourpulse of [³H]-leucine (4.5 μCi/ml) was given before cells were collectedonto filters with a Skatron harvester. Samples were counted in a Beckmanscintillation counter. Each experiment was performed in 4 replicates.Results were calculated into a mean value, and recorded as a percentageof control cells.

Serum antibody detection.

Anti-DT antibodies were detected in human serum by ELISA. CRM9 (10μg/ml) was adsorbed to Costar 96-well EIA/RIA flat bottom plates(Costar, Cambridge, Mass., USA) for 2 hours and then washed in phosphatebuffered saline (PBS) containing 0.1% Tween 20. Each well was thenincubated with PBS containing 3% gelatin to prevent non-specific bindingof antibodies to the plastic. Serum samples were diluted in PBScontaining 0.1% Tween 20 and 0.3% gelatin prior to addition to theplate. After 1 hour incubation, the wells were washed as above, andincubated for an additional hour with protein A/G-alkaline phosphatase(1:5,000; Pierce, Rockford, Ill., USA). Wells were washed, andphosphatase substrate (Pierce) was added following the manufacturer'sdirections. After 30 minutes color development was stopped with NaOH andthe optical density (OD) was measured with a kinetic microplate reader(Molecular Devices Corporation, Palo Alto, Calif., USA). Each sample wasperformed in triplicate. Results are presented as O.D. values andantibody titers.

Endocytosis assay.

UCHT1-CRM9 was iodinated using the Bolton-Hunter reagent (NEN Dupont,Wilmington, Del., USA) as described (16). Jurkat cells were washed twicewith binding medium (RPMI 1640 supplemented with 0.2% bovine serumalbumin, 10 mM Hepes (pH 7.4) and without sodium bicarbonate). Cells(1.5×10⁶) were incubated for 2 hours on ice with ¹²⁵I-UCHT1-CRM9 (1×10⁻⁹M) that had been pre-incubated with serum or binding medium. Unboundantibody was removed by washing the cells twice in PBS (pH 7.4) withcentrifugation and resuspension. Duplicate samples were incubated for 30minutes on ice or at 37° C. One sample from each temperature point wascentrifuged at 800 x g to separate the total cell associated (pellet)from the exocytosed or dissociated counts (supernatant). Both fractionswere counted in a Beckman a γ-counter. To determine the amount ofinternalized immunotoxin, cells from the second sample at eachtemperature were incubated in low pH medium (binding medium containing10 mM morpholinoethanesulfonic acid, all of which was titrated to pH 2.0with HCl) for 5 minutes to dissociate the surface bound ¹²⁵I-immunotoxin(17). Samples were centrifuged at 800 x g to separate the internalized(pellet) from the membrane bound (supernatant). Both fractions werecounted in a Beckman γ-counter (Beckman, Fullerton, Calif., USA).

Serum with anti-DT antibodies inhibits UCHT1-CRM9 toxicity.

Since humans are immunized against DT, the presence of anti-DTantibodies in the serum was determined by ELISA (Table 3). In a limitedsample population, 80% of the serum samples had an anti-DT antibodytiter of 1:100 or above. The vaccination status of the donors was notavailable. To determine the effect of these antibodies on UCHT1-CRM9toxicity, the immunotoxin was pre-incubated with differentconcentrations of serum and the toxicity of the mixture was assayed(Table 3). Serum samples without a significant ELISA O.D. (2 fold abovebackground) were incapable of affecting UCHT1-CRM9 toxicity at highconcentrations of serum (1:10). However, serum samples with a positiveELISA result could neutralize the cytotoxic effect at 1:10 dilution, andthose with a high ELISA O.D. (7-11 fold above background) inhibitedtoxicity even at a 1:100 dilution. Similar results were seen in assaysconducted with monkey serum samples.

TABLE 3 Human serum with anti-DT antibodies inhibits the toxicity ofUCHT1- CRM9 and the inhibition correlates with the anti-DT titer ELISAProtein Synthesis^(b) (% control) Sample O.C. (X ± S.D.) Titer 1:101:100 1:1,000 10010 0.738 ± 0.017 1:750 97 ± 3 79 ± 8  2 ± 0 10011 0.568± 0.048 1:500 104 ±  13 ± 2  2 ± 0 10012 0.491 ± 0.025 ND^(c) 96 ± 3 19± 2  2 ± 0 10013 0.411 ± 0.052 1:500 105 ± 8  7 ± 1 2 ± 0 10014 0.390 ±0.047 1:500 96 ± 2 7 ± 6 2 ± 0 10015 0.353 ± 0.008 1:250 125 ± 6  6 ± 42 ± 0 10019 0.359 ± 0.019 1:250 101 ± 7  6 ± 1 2 ± 0 10016 0.141 ± 0.0151:100 22 ± 1 3 ± 0 2 ± 0 10017 0.100 ± 0.006 <1:100  4 ± 0 3 ± 0 2 ± 010018 0.071 ± 0.001 <1:100  2 ± 0 2 ± 0 2 ± 0 Goat 1.450 ± 0.013 1:10⁵102 ± 19  104 ± 3  ^(a)ELISA was performed in triplicate for each serumsample as described under “Materials and Methods.” The O.D. values werederived from 1:100 dilutions and presented as a mean value ± SD. Thebackground value was 0.060 ± 0.02. titers are recorded as the highestserum dilution that showed a positive reaction in ELISA. ^(b)UCHT1-CRM9(2 × 10⁻¹⁰) was incubated with different dilutions of serum for 30 min.The mixture was then added to cells as described under “Materials andMethods.” Four replicates were performed for each sample. Data arepresented as a mean value ± s.c. in percentage of the control counts.UCHT1-CRM9 inhibited protein synthesis to 2.0% of controls. The goatanti-DT serum could be diluted to 1:10,000 and still completelyinhibited the toxicity of UCHT1-CRM9. ^(c)ND, not doneSera do not inhibit endocytosis of UCHT1-CRM9.

The inhibitory effect of serum on UCHT1-CRM9 toxicity could be due toprevention of the immunotoxin binding to the cell surface or theendocytosis of UCHT1-CRM9 into the cell. Endocytosis assays wereconducted using ¹²⁵I-UCHT1-CRM9 to determine if either of theseprocesses were affected by anti-DT antibodies present in sera. Theresults indicate that the presence of serum (goat anti-DT or human)reduces as much as 80% of the immunotoxin counts binding to the cellsurface (Table 4). While this is a significant reduction in binding,limiting 90% of input immunotoxin (one log less UCHT1-CRM9) in toxicityassays reduces protein synthesis to <25% of controls (see FIG. 3). Incontrast, the inhibitory effect of serum containing anti-DT antibodiesis 100%. Therefore the effect of the anti-DT antibodies is not all atthe level of inhibition of binding to the cell surface. Thepre-incubation of ¹²⁵I-UCHT1-CRM9 for 2 hours on ice and subsequentwashing at room temperature resulted in 18 to 25% of the total cellassociated counts internalized (Table 4). After incubation for 30minutes at 37° C., there is a doubling of internalized counts both withand without serum, indicating that the same percentage of labeledimmunotoxin is endocytosed. The identical dilutions of serum wereincubated with non-labeled UCHT1-CRM9 and used in protein synthesisinhibition assays. The results demonstrate that the ratio of immunotoxinto serum used was capable of completely inhibiting the toxicity (Table4), although the endocytosis of UCHT1-CRM9 was not affected.

TABLE 4 Inhibition of UCHT1-CRM9 toxicity by serum does not correlatewith inhibition of endocytosis. Serum Time % of Bound Protein SynthesisSample (37° C.) % Bound internalized (% Control) — 0 100 23.6. N.D.^(a)— 30 100 58.8 3 ± 1 Human 0 20 18.1 N.D.^(a) Human 30 19 35.9 105 ± 5  —0 100 25.3 N.D.^(a) — 30 100 54.0 3 ± 1 Goat 0 37 24.4 N.D.^(a) Goat 3033 50.7 92 ± 14 [¹²⁵I]-UCHT1-CRM9 (2 × 10⁻⁹ M) was incubated with mediumor anti-DT serum (1:4 dilution of human sample 10010 or a 1:1,000dilution of goat serum; Table 3) for 30 minutes at room temperature.This mixture was added to Jurkat cells (1.5 × 106) for 2 hours on ice(final concentration of [¹²⁵I]-UCHT1-CRM9 was 1 × 10-10). The cells werethen washed and endocytosis assays performed as described in Materialsand # Methods. The % Bound value represents the cell associated countsdivided by the cell associated counts divided by the cell associatedcounts without serum. Non-labeled UCHT1-CRM9 was incubated with theabove dilutions of sera and the resulting mixture was used in proteinsynthesis inhibition assays. the results shown are representative of twoindependent assays. n.d.: not done.The inhibitory effect of anti-DT antibodies can be removed byadsorption.

To prevent the inhibitory effect of serum as well as gain insight intothe mechanism by which serum inhibits toxicity, experiments weredesigned to adsorb the protective anti-DT antibodies from the serum. Theserum (a pool of all human sera with positive anti-DT ELISA or goatanti-DT) was pre-incubated for 30 minutes with increasing concentrationsof CRM197 (an A-chain mutant of DT with no enzymatic activity), MSPΔ5 (atruncation mutant missing the last 150 amino acids) and the purified A-and B-subfragments of DT (FIG. 2A). The adsorbed serum was thenincubated with UCHT1-CRM9 in protein synthesis inhibition assays.CRM197, the full length DT-like construct, was capable of completelyadsorbing the protective antibodies from both goat (FIG. 2B) and pooledhuman serum (FIG. 2C). The B-subfragment of DT is also capable ofcomplete adsorption, however ˜100 fold more is required. TheA-subfragment of DT had little or no effect on either serum, althoughthe serum samples were demonstrated to contain antibodies reactive toboth the A- and the B-subfragments by Western Blot analysis. Of interestwere the results seen with MSPΔ5, the truncation mutant. Adsorption ofgoat serum with MSPΔ5 gave a dose dependent removal of the serum'sprotecting effect (FIG. 2B). However, this adsorption could not bringtoxicity down to levels obtained when CRM197 or the B-subfragment wasused.

In contrast to the results observed with the goat serum, MSPΔ5 hadlittle effect on pooled human serum (FIG. 2C). These results suggestthat the pre-existing anti-DT antibodies important for the protectingeffect in human serum are mainly directed against the last 150 aminoacids of DT.

sFv-DT390 is not inhibited by anti-DT antibodies present in human sera.

Having observed that the epitope(s) recognized by the antibodiesimportant for protection lay in the C-terminal 150 amino acids, asingle-chain immunotoxin was generated with the first 390 amino acids(out of 535) of DT. Position 390 was chosen for 2 reasons: first, the 3dimensional structure of DT suggested that this position was an externalpoint on the molecule away from the enzymatic domain (18), and second,fusion toxins have been generated with longer DT subfragments with noreports of serum effects (19). The DNA encoding the first 390 aminoacids of DT was ligated to DNA encoding the anti-CD3εsFv (V_(L) linkedto V_(H) using a (Gly₃Ser)₄ linker sequence). The predicted molecularweight for the fusion protein is 71,000 Daltons and has been confirmedby Western Blot analysis of both in vitro transcribed and translatedprotein as well as protein isolated from E. coli using goat anti-DTantibodies. The toxicity of sFv-DT390 protein, isolated from E. colistrain BL21/DE3, was compared to UCHT1-CRM9 in protein synthesisinhibition assays (FIG. 3A). The IC₅₀ (concentration required to inhibitprotein synthesis to 50% of controls) of sFv-DT390 was 4.8×10⁻¹¹ Mcompared to 2.9×10⁻¹² M for UCHT1-CRM9, a 16-fold difference. Todemonstrate the specificity of the sFv-DT390 construct, competitionexperiments were performed using increasing concentrations of UCHT1antibody as competitor (FIG. 3B). The results showed that approximately⅛ antibody is needed to compete the sFv-DT390 toxicity to 50% ascompared to UCHT1-CRM9. The antibody was capable of totally competingtoxicity of both constructs thereby showing their specificity. Theimmunotoxins were then subjected to protein synthesis assays in thepresence of increasing dilutions of serum (Table 5).

UCHT1-CRM9 toxicity was completely inhibited with a 1:10 dilution of thehuman sera but at a 1:100 dilution toxicity was equivalent to controlswithout serum. In contrast, the sFv-DT390 immunotoxin is only partiallyinhibited with the 1:10 dilution of the human sera and the 1:100dilution no effect on the toxicity. Both immunotoxins are completelyinhibited by goat anti-DT serum (1:1,000 dilution). These resultsindicate that the sFv-DT390 immunotoxin partially evades thepre-existing anti-DT antibodies present in most human sera.

These results indicate that the pre-existing anti-DT antibodies presentin human serum inhibit the toxicity of the immunotoxin UCHT1-CRM9. Thisinhibition of toxicity was also observed with goat anti-DT serum,however less goat serum was needed to completely inhibit toxicity. Theexperiments were designed in such a way to mimic the in vivo situation.The peak concentration of circulating immunotoxin currently being testedin animal models is 1×10⁻⁹ M. The immunotoxin concentration incubatedwith the 1:10 dilution of human serum was 1×10⁻¹⁰ M, thus approximatingin vivo conditions. The inhibition of toxicity correlates with the serumantibody levels as determined by ELISA (Table 4), indicating that serawith higher anti-DT titers have a stronger inhibitory effect. Similarly,the goat anti-DT serum which gave the highest ELISA value could bediluted 10,000 times and still completely inhibited UCHT1-CRM9 toxicity.Since this correlation exists, there is no indication that any othercomponent of the serum inhibits the-toxicity of UCHT1-CRM9.

Furthermore, the data show that a titer of 1:100 dilution is necessaryfor an inhibition of the immunotoxin toxicity. A construct in which thefirst 486 amino acids of DT were fused to interleukin-2, DAB₄₈₆IL-², wasused in lymphoid malignancy patients. A partial response to DAB₄₈₆IL-2was observed in several patients who had a anti-DT titer below 1:100dilution prior to the treatment.

Intoxication of cells by immunotoxins can be subdivided into fourgeneral stages: 1) specific binding to the cell surface, 2) endocytosisinto the cell, 3) translocation of enzymatic domain of the toxin out ofthe endosome and 4) enzymatic inactivation of the target molecule. Theresults presented indicate that, while the amount of immunotoxinreaching the cell surface is lower in the presence of serum, the samepercentage of bound immunotoxin is endocytosed. Taking into account thereduced amount of immunotoxin bound to the cell, the amount ofendocytosed immunotoxin should intoxicate the cells to below 25% ofcontrols. However, the immunotoxin had no effect on protein synthesis inthe presence of serum containing anti-DT antibodies. Since theA-subfragment of DT could not adsorb the protective effect of serumwhile the B-subfragment could, the effect of serum is not likely to beat the level of inhibiting enzymatic activity of the toxin. Therefore,the anti-DT antibodies probably affect the translocation of theA-subfragment into the cytosol.

CRM197, B-subfragment, and MSPΔ5 could adsorb the protecting anti-DTantibodies from the goat and rhesus monkey sera. However, among the 3 DTmutants, MSPA5 could not prevent the UCHT1-CRM9 toxicity in the presenceof the human sera, showing a difference in the anti-DT antibodyrepertoire among humans, goat and-rhesus monkeys. This difference doesnot seem to be due to immunization routes, because monkeys used in thepresent study were not immunized for DT and presumably acquire theantibodies after a natural infection with toxigenic strains of C.diphtheriae. There have been reports showing that rhesus monkeys andhumans shared a similar antibody repertoire (21), but the presentresults suggest that the effect of antibodies from the host for whomimmunotoxin treatment is intended should be useful.

To overcome the blocking effect of the pre-existing anti-DT antibodiesin human sera, there are basically two pathways existing. One is toneutralize the antibodies with non-toxic DT mutants, and the other is tomodify the DT structure used for making immunotoxin (3). The antibodyneutralization pathway has been tested in monkey studies of FN18-CRM9treatment as described above.

The present results showed that although antibodies against both A- andB-subfragments existed in human sera, MSP5 could not neutralize thepre-existing protective anti-DT antibodies, and therefore could notprevent the inhibition of the cytotoxicity of UCHT1-CRM9. However, itdid block the inhibitory effect of the goat and monkey sera. Thisprompted the construction of the present recombinant immunotoxin,sFv-DT390. The IC₅₀ of sFv-DT390 is 4.8×10⁻¹¹ M, 1/16 as potent asUCHT1-CRM9. Like many other single-chain constructs, sFv-DT390 ismonovalent as compared to immunotoxins generated with full length,bivalent antibodies. The reduced toxicity in sFv-DT390 could beexplained primarily on this affinity difference. Immunotoxins generatedwith purified F(ab)′ fragments of antibodies also show an in vitro lossin toxicity (generally a 1.5 log difference) when compared to theircounterparts generated with full length antibodies (22). The toxicity ofsFv-DT390 is comparable to that reported for DAB486IL-2 (23). From thepresent data some advantages of sFv-DT390 are expected. First, sFv-DT390is only ⅓ of the molecular weight of UCHT1-CRM9. The molar concentrationof sFv-DT390 will be 3 times higher than that of UCHT1-CRM9 if the sameamount is given (for example, 0.2 mg/kg). Therefore, their difference inpotency could be reduced to approximately 5 times. Second, in an invitro experiment (Table 5), the same molar concentration of sFv-DT390and UCHT1-CRM9 was used for serum inhibition test, although the formeris only 1/16-potent compared to the latter. The pre-existing anti-DTantibodies in human sera could only partially block the toxicity ofsFv-DT390 while the effect of UCHT1-CRM9 was completely blocked. Thus,sFv-DT390 is expected to bypass the anti-DT antibodies in in vivosituations while UCHT1-CRM9 cannot. Third, sFv-DT390 contains only thevariable region of UCHT1, and is expected to have less immunogenicity inhuman anti-mouse antibody (HAMA) responses than the native murineantibody UCHT1. Finally, the production cost of sFv-DT390 is much lowerthan that of UCHT1-CRM9. Based on these reasons, sFv-DT390, or otherswith similar properties, are expected to be useful in the treatment ofT-cell mediated diseases in humans, especially in anti-DT positiveindividuals and in patients who need repeated treatments. To obtainevidence supporting this assumption, it is only necessary to construct arhesus monkey analog of sFv-DT390, and test it in monkey models asdescribed in previous examples.

TABLE 5 Anti-DT antibodies present in human sera have reduced effect onsFv-DT390 toxicity. Protein synthesis (% Control) ELISA value UchT1CRM9sFv-DT390 Serum Sample (± S.D.) 1:10 1:10² 1:10³ 1:10 1:10² 1:10³ 100120.491 ± 0.025 119 ± 24 8 ± 2 ND^(a) 47 ± 9 21 ± 8 ND Pooled 0.331 ±0.015 108 ± 37 7 ± 1 ND^(a) 49 ± 7 16 ± 7 ND Goat 1.450 ± 0.013 ND ND 94± 21 ND ND 8 ± 11 *Not done UCHT1CRM9 or sFv-DT390 (2 × 10⁻⁹ M) wasincubated with the indicated dilutions of serum for 30 min. The mixturewas then added to cells as described under “Materials and Methods.” Thefinal concentration of immunotoxin on cells was 1 × 10⁻¹⁰ M. Fourreplicates were performed for each sample. Data are presented as a meanvalue ± S.D. in percentage of the control counts. UCHT1-CRM9 inhibitedprotein synthesis to 5% of controls while the sFv-DT390 # inhibitedprotein synthesis to 18% of controls. The ELISA value was determinedusing a 1:100 dilution of serum. The results are representative of twoindependent experiments.

EXAMPLE 10 Expression and Characterization of A Divalent ChimericAnti-human CD3 Single Chain Antibody

Murine anti-CD3 monoclonal antibodies (mAbs) are used in clinicalpractice for immunosuppression. However, there are two major drawbacksof this treatment: the associated cytokine release syndrome and humananti-mouse antibody response. To overcome these side effects, a chimericanti-human CD3 single chain antibody, scUCHT1 was generated. It is anIgM variant of the UCHT1 described in Example 9. scUCHT1 consists of thelight and heavy variable chain binding domains of UCHT1 and a human IgMFc region (CH₂ to CH₄). The method used was reported by Shu et al. [37]and is further described below. The following data show that theengineered chimeric anti-CD3 single chain antibody (scUCHT1) will beuseful in clinical immunosuppressive treatment.

Oligonucleotide primers and DNA amplification.

Primers used for the antibody engineering are listed in Table 6, and theprimer sequences are based on published data [13]. The procedures ofcloning scUCHT1 is schematically depicted in FIG. 4. mRNA isolated fromUCHT1 hybridoma cells (provided by Dr. P. C. Beverley, Imperial CancerResearch Fund, London was reverse transcribed into cDNA. The V_(L) andV_(H) regions of UCHT1 were amplified with polymerase chain reaction(PCR) from the cDNA using primer pairs P1, P2 and P3, P4 respectively.Primers P2 and P3 have a 25 bp complementary overlap and each encoded apart of a linker peptide (Gly₄Ser)₃. The single chain variable fragment(V_(L)-linker-V_(H)) was created by recombinant amplification Of V_(L)and V_(H) using primers P1 and P4. A mouse kappa chain signal sequencewas added at the V_(L) 5′-end by PCR, first with primers SP2 and P4, andthen with primers SP1 and P4. The human IqM Fc region (CH₂ to CH₄) wasamplified from the plasmid pBlue-huIgM (kindly provided by Dr. S. V. S.Kashmiri, National Cancer Institute, Bethesda. This gene fragment wasabout 1.8 kb. The V_(L)-linker-_(VH)-CH² region which is important forantigen recognition was confirmed by sequence analysis. Finally, thesingle chain variable fragment and the human IgM Fc region were clonedinto plasmid pBK/CMV (Stratagene, La Jolla, Calif., USA). Using thegenerated pBK/scUCHT1 plasmid as template, an in vitrotranscription-translation assay yielded a product of 75 kDa, theexpected size.

TABLE 6 Sequences of oligonucleotide primers used for PCR amplificationSequence ID Sequence Number 5′ 3′ Primers RE sites SEQ ID NO. 7GACATCCAGATGACCCAGACC P1 (UCHT1 VL5) SEQ ID NO. 8CCTCCCGAGCCACCGCCTCCGCTGCCTCCGCCTCCTTTTA P2 (UCHT1 VL3)TCTCCAGCTTG(T)GTC(G)CC SEQ ID NO. 9GCAGCGGAGGCGGTGGCTCGGGAGGGGGAGGCTCGGAGGT P3 (UCHT1 VL5) GCAGCTTCAGCAGTCTSEQ ID NO. 10 GC AAGCTT GAAGACTGTGAGAGTGGTGCCTTG P4 (UCHT1 VH3) Hind IIISEQ ID NO. 11 GTCTCTTCAAAGCTT ATTGCC(T)GAGCTGCCTCCCAAA P5 (HuIgM-CH2)Hind III SEQ ID NO. 12 GCA TCTAGA TCAGTAGCAGGTGCCAGCTGTGT P6 (HuIgM-CH4)Xba I SEQ ID NO. 13 CG GTCGAC ACCATGGAGACAGACACACTCCTGTTATGGGT SP1(signal seq1) Sal I ACTGCTGCTCTGGGTTCCA SEQ ID NO. 14GTACTGCTGCTCTGGGTTCCAGGTTCCACTGGGGACATCC SP2 (signal seq2) AGATGACCCAGRE: restriction enzyme. Restriction sites appeared in the primers wereunderlined and bold. The primers listed as SEQ ID NO: 8 and SEQ ID NO:11 consisted of a mixture of the sequence without the nucleotide(s) inparentheses and the sequence (s) with the nucleotide(s) in parenthesesreplacing the immediately preceding nucleotide(s) in the sequence.Expression in COS-7 and SP2/0 cells.

The gene fragment encoding scUCHT1 was then cloned into an expressionvector pLNCX [36]. The scUCHT1 gene construct was introduced into COS-7cells with a calcium-phosphate method [32], and introduced into SP2/0myeloma cells by electroporation [33]. Cells transfected were selectedwith 500 μg/ml G418 (GIBCO/BRL, Gaithersburg, Md., USA) in DMEM medium.The drug resistant transfectants were screened for scUCHT1 secretion byan anti-human IgM ELISA technique. Transfectants secreting scUCHT1 werecloned by limiting dilution.

Two stable clones, COS-4C10 and SP2/0-7C8, which could produce about 0.5mg/ml scUCHT1 in culture medium, were selected for further evaluation.The culture supernatant of COS-4C10 and SP2/0-7C8 cells was analyzed byimmunoblotting using anti-human IgM antibody (FIG. 5). Human IgMantibody was included as a control in the analysis. Under reducingconditions, scUCHT1 produced by COS-7 and SP2/0 cells had a similarelectrophoretic mobility to that of the control human IgM heavy chain(75 kDa). Under non-reducing conditions, scUCHT1 from COS-7 cellsappeared as a single band of approximately 150 kDa, which was thought tobe a homodimer of the single chain antibody. SP2/0 cells mainly produceda protein of similar size with some higher molecular weight products.

In constructing scUCHT1, the domain orientation of sFv, V_(H)-V_(L),which Shu et al. used to V_(L)-V_(H) orientation, was changed so thatthe heavy chain constant domains were linked to the V_(H) domain. Inmammalian cells, secretion of immunoglobulin molecules is mediated bylight chain, and free light chain is readily secreted [38]. However,free heavy chain is generally not secreted [39]. In a bacterialexpression system, the yield of secreted sFv with a V_(L)-V_(H) domainorientation was about 20-fold more than that obtained with a V_(H)-V_(L)domain orientation [40]. It was reasoned that V_(L) at the NH₂-terminalposition and V_(H) linked to heavy chain constant region in scUCHT1construct might enhance the secretion of this immunoglobulin-likemolecule in mammalian cells. In fact scUCHT1 was efficiently produced byboth COS-7 and SP2/0 cells. Hollow fiber culture should increase itsproduction. Moreover, scUCHT1, the IgM-like molecule, has a secretorytailpiece with a penultimate cysteine (Cys 575) which is involved inpolymerization and also provides retention and degradation of IgMmonomers [41-43]. Replacing the Cys 575 with serine might also greatlyimprove the yield.

scUCHT1 secreted from COS-7 cells was shown to be a divalent form byimmunoblotting, suggesting a disulfide bond linkage of two monovalentmolecules. The disulfide bond is likely situated between the CH2 and CH3regions, where the Cys 337-Cys 337 disulfide bond is thought to exist.Cys 337 is believed to be sufficient for assembly of IgM monomers, andwas neither sufficient nor necessary for formation of polymers. However,Cys 575 was necessary for assembly of IgM polymers, and Cys 414 was notrequired for formation of IgM monomers or polymers [44]. This divalentform of the single chain antibody should increase its binding affinity.While scUCHT1 produced from SP2/0 cells was mainly in the divalent form,a small fraction of the antibody had a higher molecular weight, nearlycomparable to that of the human IgM pentamer, the natural form ofsecreted human IgM.

Western blotting analysis of scUCHT1.

scUCHT1 was precipitated from the culture supernatant using goatanti-human IgM-Agarose (Sigma, St. Louis, Mo., USA), and separated on4-20% SDS-PAGE gradient gel under reducing and non-reducing conditions.The separated proteins were transferred to ProBlottTM membrane (AppliedBiosystems, Foster City, Calif., USA) by electroblotting at 50 volts for1 hour. The membrane was blocked and incubated with alkaline phosphataselabeled goat anti-human IgM antibody (PIERCE, Rockford, Ill., USA)following the manufacturer's instruction. Color development was carriedout with substrate NBT/BCIP (PIERCE).

Purification of scUCHT1.

Culture supernatant was mixed with anti-human IgM-Agarose, and incubatedat 4° C. with shaking overnight, and then the mixture was transferred toa column. The column was washed with washing buffer (0.01 MNa-phosphate, pH 7.2, 0.5 M NaCl) until the OD280 of flow-through was<0.01. scUCHT1 was eluted with elution buffer (0.1 M glycine, pH 2.4,and 0.15 M NaCl). The fractions were neutralized with 1 M Na-phosphate(pH 8.0) immediately, and then concentrated and dialyzed against PBS.

Competitive binding assay.

The parental antibody UCHT1 was iodinated using Bolton-Hunter Reagent(NEN, Wilmington, Del., USA) as described previously [34]. The¹²⁵I-labeled UCHT1 was used as tracer and diluted with DMEM medium to0.3-0.6 nM. UCHT1 and the purified scUCHT1 from COS-7 and SP2/0transfectant cells were used as competitors. Human CD3 expressing Jurkatcells were suspended in DMEM medium (2×10⁷/ml). 50 μl of such cellsuspension (1×10⁶) was incubated with 50 μl diluted tracer and 50 mldiluted competitors on ice for 2 hours. Afterwards, cells were pelleted,and counted in a gamma counter. Results were expressed as a percentageof the ¹²⁵I-UCHT1 bound to cells in the absence of competitors (FIG. 6).

scUCHT1 from both COS-7 and SP2/0 cells could specifically inhibit thebinding of ¹²⁵I-UCHT1 to Jurkat cells in a dose dependent way. As theconcentration of the competitors (UCHT1, scUCHT1 from COS-7 and SP2/0cells) increased from 1 to 100 nM , the tracer (¹²⁵I iodinated UCHT1)bound to Jurkat cells decreased from 80% to nearly 0%. No significantdifference was observed among the affinity curves of UCHT1 and scUCHT1from COS-7 and SP2/0 cells. This indicates that the engineered antibodyscUCHT1 has nearly the same affinity as UCHT1. Moreover, scUCHT1contains human IgM constant region, and is expected be less immunogenicthan UCHT1. The degree of its immunogenicity might vary due to themurine variable region of scUCHT1. Humanized variable regions byCDR-grafting or human variable regions can be used to further reduce itsimmunogenicity [31].

T-cell proliferation assay.

T-cell proliferation in response to UCHT1 and scUCHT1 was tested onhuman PBMCs from a healthy donor (FIG. 7). Human peripheral bloodmononuclear cells (PBMCs) were isolated from blood of a healthy adult bydensity centrifuge over Ficoll-Hypaque gradient [34]. The PEMCs wereresuspended in RPMI 1640 supplemented with 10% FCS and aliquoted to96-well U-bottom plates at 5×10⁴ cells/well. Increasing amounts ofanti-CD3 antibodies (UCHT1, scUCHT1) were added. After 72 hours ofculture at 37° C. in a humidified atmosphere containing 5% CO₂, 1 μCi[³H]thymidine (NEN) was added to each well. 16 hours later, cells wereharvested and [³H]thymidine incorporation was counted in a liquidscintillation counter.

The parental antibody UCHT1 started to induce proliferation at 0.1ng/ml, and peaked at 100 ng/ml. A small drop in CPM was observed as theconcentration increased to 1,000 ng/ml. However, [³H]thymidineincorporation in PBMCs incubated with scUCHT1 was only slightlyincreased in the range of 0.1-10 ng/ml, and when the concentration washigher than 10 ng/ml, the incorporated counts decreased and were closeto 0 counts at 1,000 ng/ml.

Measurement of TNF-α and IFN-γ.

TNF-α and IFN-γ productions of human PBMCs induced by UCHT1 and scUCHT1were measured with ELISA. 4×10⁵ PBMCs were cultured with serialdilutions of anti-CD3 antibodies (UCHT1, scUCHT1) in 96-well flat-bottomplates in RPMI 1640 supplemented with 10% FCS. Supernatant was collectedat 36 hours for TNF-α and 72 hours for IFN-γ after the start of theculture (351. TNF-α and IFN-γ were measured with ELISA kits (EndogenInc. Cambridge, Mass., USA) following the manufacturer's instruction.

The native antibody UCHT1 induced production of both TNF-α and IFN-γ ina dose dependent way. (FIG. 8 a and 8 b). Higher concentration of UCHT1induced higher production of TNF-α and IFN-γ. On the contrary, scUCHT1did not induce secretion of TNF-α at any concentration (FIG. 8 a), andinhibited IFN-γ production when its concentration was higher than 0.1ng/ml (FIG. 8 b). At the time of supernatant harvesting, the PBMCscultured with UCHT1 and scUCHT1 were also checked with trypan blueexclusion test. Cells were shown to be alive in both situations. InTNF-α and IFN-γ ELISA assays, an unrelated human IgM was included and itdid not affect the TNF-a and IFN-g production.

Anti-CD3 mAbs can induce T cell activation and proliferation both in invitro and in vivo situations [45]. Crossing-linking of anti-CD3 antibodybetween T cells and FcR expressing cells is an essential step in thisprocess [46]. T cell activation therefore reflects an efficientinteraction of the mAb with a human FcR. Previous data of in vitro studyindicated that T cell activation resulted in increased production ofTNF-α, IFN-γ, and IL-2 [24]. Human IgG Fc receptors (FcγR I, FcγR II,FcγR III) are distributed on human monocytes, T, B lymphocytes, and NKcells [47]. FcγR I and FcγR II can recognize both mouse and human IgG.In accordance with the above observation, UCHT1 was potent in inductionof T cell proliferation and TNF-α and IFN-γ release. Human IgM Fcreceptor (FcμR) was reported to be present mainly on a small fraction ofB lymphocytes, NK cells, and possibly a helper subset of T lymphocytes[47, 48]. Pentamer form of IgM and an intact CH₃ domain are required foroptimal binding to FcμR. Monomeric or dimeric subunits of IgM are lessefficient in binding to FcγR [49, 50]. Cross-linking of IgM to FcμR on Tcells inhibited the mitogen-induced T cell proliferation, and FcμR mayfunction as a negative signal transducing molecule [51, 52].

Therefore, it can specifically bind to human CD3 molecule and FcμR. Itis conceivable that scUCHT1 can cross-link human B and T cells, andpossibly T and T cells. In an in vitro assay, scUCHT1 from both COS-7and SP2/0 cells had little effect in the T cell proliferation assay atlow concentrations (below 10 ng/ml), and became inhibitory as theconcentration increased. In accordance with these results, scUCHT1 didnot induce TNF-α production and even inhibited the basal yield of IFN-γ.

The present chimeric anti-CD3 single chain antibody scUCHT1 possesseshigh human CD3 binding specificity and affinity, and does not induce Tcell proliferation and cytokine release. Moreover, it has a human IgM Fcfragment, which should decrease the possibility of inducing humananti-mouse antibody response. Thus, scUCHT1 can be used for clinicalimmunosuppressive treatment.

EXAMPLE 11 Cloning the full-length of DT gene for the construction ofDTM2.

Corynebacteriophage beta (C. diphtheriae) tox 228 gene sequence was fromgenebank. (Science 221, 885-858, 1983). The sequence is 2220 bp. Thereare 300 bp of 5′ untranslated region (1 to 300) including the promotersequence around (−180 to −10), 1682 of coding region (301-1983)including signal peptide (301 to 376), A chain (377 to 955) and B chain(956 to 1983), and 3′ untranslated region (1984 to 2220).

The full-length DT was amplified in two fragments. The pelB leadersequence (ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTACTGCGCT GCC CAA CCA GCG ATG GCC 3′) SEQ ID NO:1) was added to the 5′ endof the DT coding sequence to all the constructs during polymerase chainreaction by primer EcosignalDT-1 and EcosignalDT-2. The upstreamfragment of 311 bp (from position 301 to 546 bp) was amplified by oligoEcosignalDT-2 and p546R with CRM9 DNA as a template and the downstreamfragment of 1471 bp was amplified by p514S and p1983R with the DTM1 DNAas template. Then, the combined PCR product of full-lenth DT wasamplified with primer EcosignalDT-1 and p1983R. As a result, theamplified DT coding sequence (position 376 to 1983bp) acquired the pelBleader sequence added to the 5′ end and contains the two mutant sites[(508 Ser to Phe) and (525 Ser to Phe)] as DTM1 does.

Primers:

EcosignalDT-1 5′ ATG AAA TAC CTATTG CCT ACG GCA GCC GCT GGA TTG TTA TTACTC GCT GCC CAA 3′ (SEQ ID NO:2)

EcosignalDT-2 5′ GGA TTG TTA TTA CTC GCT GCC CAA CAA GCG ATG GCCGGC GCTGAT GATGTT GTT GAT TC 3′ (SEQ ID NO:3) p546R: 5′CGGTACTATAAAACTCTTTCCAATCATCGTC 3′ (SEQ ID NO:4) p514S: 5′GACGATGATTGGAAAGAGTTTTATAGTACCG 3′ (SEQ ID NO:5) p1983R:5′AGATCTGTCGA/CTCATCAGCTTTTGATTTCAAAAAATAGCG 3′ (SEQ ID NO:6).

A mutant residue was introduced at position 52. The glycine (GGG) atposition 52 wild type DT was substituted by glutamic acid (GAG). The twoprimers p546R and p514S carried the mutant codon (GGG to GAG). The PCRproducts of these two primers contained the substituted codon (GAG)instead of codon GGG. The jointed double stranded DNA of the twofragments (1683bp) were cloned into pET 17b by restriction site NdeI andBamHI.

The data show that anti-human blocking antibodies are specificallydirected at the toxin C-terminus. Although a specific sequence derivedfrom the UCHT1 VLV_(H) regions is described, anyone skilled in the artcould make sequence variations in VLVH domains which can be designed toincrease the affinity of the sc-anti-CD3-antibody conferring a morefavorable therapeutic ratio to fusion immunotoxins using thisderivative. Such modifications are within the scope of the presentteaching. The disadvantage of the monovalent antibody VLVH construct, isthat it has a lower affinity for T cells compared to the chemicallycoupled conjugate which utilizes a divalent antibody.

These are believed to be the first instances of a sc anti-CD3antibodies. IgM was chosen since very few B cells or macrophages containIgM Fc receptors. (Binding of immunotoxin to cells other than T cellsreduces-the specificity of the anti-T cell immunotoxin and thissituation is purposefully avoided). However, using a bacterialexpression system no carbohydrate is attached to the antibody which alsoeliminates Fc receptor binding. Thus, substituting other human IgGconstant domains would be a routine modification and should be claimed.

A variety of divalent fusion protein immunotoxins-are provided. Thesehave been expressed in E. coli, and Western blots of reduced andnon-reduced SDS gels confirm that most of the immunotoxin is secreted asthe dimeric (divalent) species (FIG. 9). The position of the toxin hasbeen varied in an attempt to minimize stearic hindrance of the divalentantibody site, yet provide the best interactions with the CD3 receptorto facilitate toxin translocation across the membrane. FIG. 10 diagramsPCR amplification. FIGS. 11 and 12 show two different clones expressingdivalent immunotoxin fusion proteins cartooned in FIGS. 13 and 14,respectively. Another variation is shown in FIG. 15. The clone producingthis consists of a clone constructed by using the single chain antibodyfollowed by a stop codon and the single chain immunotoxin, all under onepromotor (Better et al. Proc. Natl'. Acad. Sci. 90:457-461, January1993). After secretion and oxidation of the interchain disulfide, 3species are present: sc divalent antibody, divalent fusion immunotoxin,and a divalent sc antibody containing only one toxin. This species isisolated by size separation and is the species cartooned in FIG. 15. Theadvantage of this species is that stearic hindrance to the divalentantibody domains is limited by the presence of only one toxin domain.Other variations are routine to construct given the methods describedherein and in the art. Those diagramed are considered to be the mostlikely to exhibit divalent character. Numerous orientations of toxinrelative to antibody domains can be made and many are expected to beeffective.

In addition, the length of the toxin C-terminus has been varied toprovide optimization between two competing functions. The numbers afterDT refer to the number of amino acid residues counting the aminoterminus of the toxin A chain as 1. The full length toxin is called DTM1and was provided by Dr. Richard Youle NINDS, NIH (Nicholls et al. J.Biol. Chem. 268(7):5302-5308, 1993). It has point mutations S to F atpositions 508 and 525. This full length toxin mutant has the essentialmutation of CRM9, S to F at 525 which reduces binding to the DT receptorby 3-4 logs without abolishing the translocation function. The othermutation S to F at 508 has been added because of previous restrictionson cloning mutant DT that can revert to wild type toxin with a minimumlethal dose of 0.1 microgram/kg by means of a single base pairreversion. Other mutations can be routinely made in the C terminus toperform this function (Shen et al. J. Biol. Chem. 269(46):29077-29084,1994). They are: F530A; K526A; N524A; V523A; K516A Y514A. A clone havinga single point mutation in DT reducing toxicity by 10-100 fold can bemade providing that the clone contains an antibody fragment fusionprotein, because chemical conjugation of antibody to DT has been shownto reduce systemic wild type toxin toxicity by 100 fold (Neville et al.J. Biol. Chem. 264(25):14653-14661, 1989). Therefore, the presentinvention provides a full length mutant DT sequence with the 525 S to Fmutation alone as well as those listed above. These same mutations arealso contemplated for the B chain mutant site in DTM2 and can be madesimilarly. Previous data with chemical conjugation has shown that thelonger the C-terminus the better the translocation function (Colombattiet al. J. Biol. Chem. 261(7):3030-3035, 1986). However, the shorter theC-terminus the less effect of circulating anti-toxin blockingantibodies. Since patients have different levels of blocking antibodieswhich can be measured (see toxicity assay in), the optimal immunotoxincan be selected for individual patients. scUCHT1 fusion proteins withDTM1 and DT483 (see FIG. 16), DT390 (FIG. 17) and DT370 (FIG. 18) havebeen cloned and expressed in E. coli. Each of these variations as wellas the divalent scUCHT1 fusion proteins using each of these toxindomains are provided.

The present invention provides an improvement on CRM197 (a non-toxictoxin mutant described in U.S. Ser. No. 08/034,509, filed Sep. 19, 1994)referred to herein as DTM2. DTM2 has the same mutation as CRM197 plustwo mutations in the C-terminus which block binding (see sheet and FIG.9). This is expected to reduce the likelihood of immune complex diseasewhich could result when CRM197 becomes bound to cells and then isfurther bound by circulating antitoxin. Kidneys are particularlysusceptible. DTM2 can not bind to cells thereby lessening thepossibility of tissue damage. In addition DTM2 is made for high levelproduction by including the pelB secretory signal for production in E.coli or a iron independent mutated promoter DT sequence cloned from CRM9DNA for production in C. diphtheriae. The essential feature of DTM2 isthe S to F mutation at 525 and the G to E mutation at 52, and aconstruct containing these two mutations is provided.

All of the constructs reported here can be expressed in E. coli usingpelB signal sequences or other appropriate signal sequences. Expressioncan also be carried out in C. diphtheriae using appropriate shuttlevectors (Serwold-Davis et al. FEMS Microbiol. Letters 66:119-14, 1990)or in protease deficient strains of B. subtilis and using appropriateshuttle vectors (Wu et al. Bio. Technol. 11:71, January 1993).

EXAMPLE 12 Thymic Injection and Tolerance Induction in Primates

Without thymic treatment, rhesus monkey renal allografts reject at amean of 7 days. Renal allografts in rhesus monkeys (age 2-5 years; 2-3kg body weight) were performed. The experimental protocol consisted offirst selecting MHC class I disparate rhesus monkey donors andrecipients. Donor lymphocytes were injected into the recipient thymusgland 7 days prior to renal allografting from the same donor. Recipientsreceived the immunotoxin of the present invention by intravenousinjection. Renal allografts were performed and recipients underwentnative nephrectomy.

Immunotoxin

Techniques for preparing anti-CD3-CRM9 (where the antibody is directedat the human T-cell receptor complex “CD3”) have previously beendescribed. See U.S. Pat. No. 5,167,956 and D. Neville et al., 89P.N.A.S. USA 2585-2589 (1992). A hybridoma secreting UCHT1 was kindlyprovided by Dr. Peter Beverly, Imperial Cancer Research Fund, and wasgrown in ascites fluid and purified over immobilized Protein A. This isan IgG1.

FN18, also an IgG1, is the rhesus analog of UCHT1 and shares with it theproperty of being a T-cell mitogen in the presence of mixed mononuclearcells. FN18 was produced in hollow fiber and purified over Protein A.The strain of C. diphtheriae used for production of CRM9, C7 (βh tox-201tox-9 h′) was obtained from R. Holmes, Uniformed Services University ofHealth Sciences, Bethesda, Md. See also V. Hu et al., 902 Biochimicia etBiophysica Acta 24-30 (1987).

Antibody-CRM9 was recovered from the supernatant of 30 literfermentation runs under careful control of iron concentration. See S. L.Welkos et al., 37 J. Virol. 936-945 (1981). CRM9 was purified bymembrane concentration, ammonium sulfate precipitation andchromatography over DEAE. See S. Carroll et al., 165 Methods InEnzymology 68 (1988).

Large scale purification of immunotoxin was accomplished by HPLC sizeexclusion chromatography on MODcol (1266 Andes Blvd., St. Louis, Mo.63132) “2×10” column packed with Zorbax (DuPont Company) GF-250 5 μm,150 Å. Fractions containing 1:1 toxin:antibody mol ratios were isolatedfor these studies.

Immunotoxins were synthesized as previously described by thiolating boththe monoclonal antibody moiety and the toxin moiety and thencrosslinking with bismaleimidohexane. See D. Neville et al., 264 J.Biol. Chem. 14653-14661 (1989). CRM9 was nicked and the monomer (Carrollet al.) was isolated by the MODcol column described above prior tothiolation.

While CRM9 is a presently preferred mutant diphtheria toxin protein,other preferred embodiments include diphtheria mutants with a mutationin the DT binding region, such as DT390, should also be suitable (as theconcept behind the immunotoxin is to replace the normal binding functionwith the antibody provided T-cell binding function, with minimalconformational change).

T-Cell Ablation.

Monoclonal antibody FN18 (specific for rhesus monkey T lymphocytes)coupled to the immunotoxin CRM9 was used to deplete peripheral bloodT-cells to levels below 200 cells /M13 in adult rhesus monkeys (measuredsix days after the injection). Some modest B cell depletion occurred.Following depletion, complete T-cell recovery takes about three to fourweeks in a juvenile rhesus monkey model using this agent. Surprisingly,notwithstanding this fast recovery, donor T-cells injected into thethymus still were not impaired in their ability to produce tolerance.

Four monkeys received 0.2 mg/kg of immunotoxin, in three divided doses(24 hours apart from each other). Another monkey received 0.133 mg/kgimmunotoxin in two divided doses (24 hours apart from each other), andthe other monkey received 0.1 mg/kg in two divided doses (24 hours apartfrom each other). Two days after the last dose of immunotoxin, allmonkeys except the last had at least 80% (actually greater than 99%)depletion of T cells both in the peripheral blood and in the lymphnodes. The lowest dose used in the last monkey reduced, but did notsubstantially eliminate either peripheral blood or lymph nodelymphocytes.

Lymphocytes

Lymphocytes to be donated are preferably pooled from axillary andcervical lymph nodes of a single donor. The nodes are harvested,strained through a mesh to separate the lymphocytes, diluted withsaline, and then injected. Alternatively, a representative “cocktail” oflymphocytes from several primates other than the donor, at least one ofwhich turns out to be the same haplotype as the likely donor, shouldalso work (if the donor is not available early enough).

Transplantation

Table 7 summarizes the outcome of renal transplants performed followingthymic injection of donor lymph node lymphocytes (mixture of T and Bcells) combined with immunotoxin therapy. Cells injected intrathymicallyconsisted of the pooled axillary and inguinal lymph node lymphocytes inthe numbers listed.

TABLE 7 Renal Allograft Survival by Treatment Group* Intrathymic Monkeyinjection FN18-CMR9 Survival (days) T4T none none 5 X9X none none 7 1FEnone none 7 H7C 10.6 × 108 none 1 donor lymphocytes W7C 9.1 × 108 none 1donor lymphocytes 93023 7.0 × 108 donor 0.2 mg/kg >517 lymphocytes92108** 1.9 × 108 donor 0.2 mg/kg 181 lymphocytes POJ 7.5 × 108 donor0.2 mg/kg >340 lymphocytes POF normal saline 0.2 mg/kg >368 PIP normalsaline 0.2 mg/kg >250 W7D none 0.2 mg/kg 51 POG none 0.2 mg/kg 84 PINnone 0.2 mg/kg >165 X3J none 0.2 mg/kg >117 *FN18-CRM9 was given on day−7, −6, −5 at a total dose of 0.2 mg/kg, i.v. Lymphocytes and salinewere injected intrathymically on day −7. ** (acute rejection 40 daysafter skin graft)

Two monkeys died of pneumonia, one at 39 days and the other at 13 days.A third monkey died at 8 days of complications stemming from a urineleak. At autopsy, none of these three monkeys had any evidence of renaltransplant rejection, either grossly or histologically.

Monkey #93023, which received the intrathymic injection and immunotoxinseven days prior to renal transplantation, had normal renal functionmore than 180 days post-transplant. A renal biopsy of his transplantedkidney at 100 days showed no evidence of rejection.

Surgical Procedures

Preferred surgical procedures include partial median sternotomy forexposure of the thymus and injection of donor lymphocytes into thethymus gland; inguinal and axillary lymphadenectomy to procure donorlymphocytes; laparotomy for procurement of the left kidney from kidneydonors; and a second laparotomy for renal transplantation and nativeright nephrectomy. All of these procedures are performed under generalanesthesia as outlined below. Serial blood draws are performed underketamine and xylazine anesthesia as outlined below.

Thymic injection is performed through a midline chest incision beginningat the sternal notch extending down to the midportion of the sternum.The sternum is divided and retracted to expose the underlying thymusgland. The thymus gland is injected with donor lymphocytes and thesternum reapproximated and the soft tissue closed.

Donor nephrectomy is performed under general anesthesia through an uppermidline incision in the abdomen. The retroperitoneal attachments of theleft kidney are divided, the ureter is ligated and divided near thebladder, and the left renal artery and vein are dissected free. The leftrenal artery and vein are ligated adjacent to the aorta and inferiorvena cava, and the kidney excised and flushed on the back table withpreservation solution.

The recipient operation for renal transplantation is performed by makinga midline abdominal incision under general anesthesia. The distal aortaand inferior vena cava are dissected free. The vena cava is clampedproximally and distally near its bifurcation and the donor renal veinanastomosed end-to-side to the recipient inferior vena cava usingrunning 7-0 proline suture. The aorta is cross-clamped proximally anddistally just proximal to its bifurcation and the donor renal arteryanastomosed end-to-side to the aorta using running 8-0 proline. Aureteroneocystostomy is then performed by making an anterior cystotomyand anastomosing the spatulated tip of the donor ureter to the bladdermucosa using B-0 proline suture. The cystotomy is then closed. Theabdomen is then closed.

Lymphadenectomy is performed through an approximately 2 cm groinincision for inguinal lymphadenectomy and a similar length incision foraxillary lymphadenectomy. The lymph nodes are excised and bleedingpoints cauterized. The skin is then closed with running 4-0 nylonsuture.

It should be appreciated that kidney transplants are merely an exampleapplication. The invention should be suitable for use with a widevariety of organs (e.g. liver, heart, lung, pancreas, pancreatic isletsand intestine).

In sum, surprisingly immunotoxins known to severely depleteT-lymphocytes will selectively deplete the host lymphocytes, withoutinterfering with the donor T lymphocytes ability to cause tolerance.Further, the extreme level of depletion caused by this immunotoxinfacilitates induction of tolerance.

EXAMPLE 13 Anti-CD3-CRM9 Immunotoxin Promotes Tolerance in Primate RenalAllografts

The ability of thymic injection and transient T lymphocyte depletion topermit development of donor-specific tolerance to rhesus monkey renalallografts was investigated. For T cell ablation, the immunotoxinFN18-CRM9, was used that depletes T cells from both the lymph node andblood compartments (see Example 5 and Neville et al. J Immunother 1996(In press)). FN18-CRM9 is composed of an anti-rhesus monkey CD3monoclonal antibody (mAb), FN18 (Neville et al., 1996), and a bindingsite mutant of diphtheria toxin, CRM9 (Neville et al. Proc Natl Acad SciUSA; 89: 2585-2589 (1992)). Compared to other anti-T cell agents used inclinical and experimental transplantation, FN18-CRM9 produces moreeffective killing of T cells, and this was the rationale for its choiceas an agent to promote transplantation tolerance. Anti-CD3-CRM9 alonesuccessfully delayed graft rejection. T cell depletion withanti-CD3-CRM9 combined with thymic injection prolonged graft survivalto >150 days in five of five recipients and induced donor-specifictolerance in four of five recipients. Donor skin grafts were acceptedlong-term, whereas third party skin grafts were promptly rejected. Theseresults are unique in their reliable induction of donor-specifictolerance as confirmed by skin grafting in a non-human primate model.This approach to tolerance reasonably correlates to induction oftolerance in humans.

MHC Typing and Donor-Recipient Selection.

Donor-recipient pairs were selected based on maximizing MHC disparity.This was based on pre-transplant cytotoxic T lymphocyte (CTL) and mixedlymphocyte reaction (MLR) analysis (Derry H, Miller R G. Fathman C G,Fitch F W, eds. New York: Academic Press, 510 (1982) and Thomas et al.Transplantation, 57:101-115 (1994)), analysis of MHC class I differencesby one-dimensional isoelectric focusing (1-D IEF) (Watkins et al. Eur JImmunol; 18:1425-1432 (1988)), and evaluation of MHC class II byPCR-based analysis.

Flow Cytometry.

Two×10⁵ lymphocytes obtained from peripheral blood or inguinal,axillary, or mesenteric lymph nodes were stained with FITC-labeled FN18or isotype control antibody. Cells were subjected to flow cytometry on aBenton Dickenson FACSCAN.

Animals and Surgical Procedures.

Outbred male juvenile rhesus monkeys (ages 1 to 3 years), virus free,were used as donors and recipients. Surgical procedures were performedunder general anesthesia, using ketamine, 7 mg/kg, i.m., and xylazine, 6mg/kg, i.m. induction, and inhalation with 1% halothane to maintaingeneral anesthesia. Post-operatively, monkeys received butorphanol, 0.25mg/kg, i.v., and aspirin, 181 mg, p.o., for pain control. Thymicinjection was performed via a limited median sternotomy to expose thethymus gland. Seven days before renal transplantation, each lobe of thethymus was injected with donor lymphocytes suspended in 0.75 to 1.0 mlnormal saline using a 27 gauge needle. Donor lymphocytes were procuredfrom the inguinal, axillary, and mesenteric lymph nodes of the donor,counted and resuspended in normal saline for injection. Heterotopicrenal transplants were performed using the donor left kidney. Followingtransplantation, the recipient underwent native nephrectomy. Graftfunction was monitored by measuring serum creatinine. Rejection wasdiagnosed by rise in serum creatinine to >0.07 mol/L, no evidence oftechnical problems, such as urine leak or obstruction at autopsy, andhistologic confirmation. Monkeys were killed with a lethal dose ofsodium pentobarbital if they rejected their kidney, and were autopsied.To test for tolerance, full thickness skin grafts were placed usingventral abdominal skin from donors placed onto the dorsal upper back ofrecipients. Grafts were evaluated daily by inspection.

Immunosuppression.

FN18-CRM9 was chemically conjugated and purified as described (Nevilleet al. 1996). It was administered intravenously at a dose of 0.2 mg/kgin 3 divided daily doses starting 7 days prior to renal transplantation.No additional immunosuppressive drugs were given to any of the monkeys,and monkeys were not isolated from environmental pathogens.

The effect of FN18-CRM9 on rhesus peripheral blood lymphocytes and lymphnode lymphocytes is summarized in FIGS. 19 a and 19 b. In addition tocausing transient T cell depletion from the peripheral blood, FN18-CRM9depleted lymph node lymphocytes almost completely at the dose given andwhen measured 0-4 days after the third dose of drug. Absolute leukocytecounts did not change significantly with treatment. Recovery times werevariable, but in general peripheral blood T lymphocytes returned towardbaseline levels 2 to 4 weeks following treatment. Recovery rates variedbetween individual monkeys.

Untreated monkeys acutely rejected their allografts (n=3) within oneweek (Table 7). Monkeys receiving lymphocytes intrathymically but noanti-CD3-CRM9 developed hyperacute rejection within 24 hours (Table 7)with the typical histologic features of hemorrhage, infarction, and adense neutrophil and lymphocyte infiltrate. Three of three recipientstreated with donor lymphocytes intrathymically and anti-CD3-CRM9 hadlong-term graft survival (Table 7). One monkey (92108) rejected itskidney 40 days after a donor and third party skin graft were placed totest for donor-specific tolerance. This monkey rejected its third partyskin graft at 10 days and a lymphocyte infiltrate in the donor skingraft developed with rejection of the renal allograft 40 days later. Theother two recipients of donor lymphocytes and anti-CD3-CRM9 weresuccessfully skin grafted from the donor with survival of these skingrafts for more than 100 days, but rejection of third party skin graftsat 10 days. All biopsies of their renal allografts showed aninterstitial infiltrate but no evidence of glomerular or tubularinfiltrates or injury. Two monkeys receiving normal saline injections inthe thymus in combination with anti-CD3-CRM9 became tolerant of theirrenal allografts. Both of these monkeys rejected a third party skingraft at 10 days and have had long-term survival of donor skin grafts.The results of all skin grafts are summarized in Table 8. Renal biopsiesof long-surviving tolerant recipients demonstrated focal interstitialmononuclear infiltrates without invasion or damage of tubules orglomeruli. Monkeys treated with anti-CD3-CRM9 alone developed laterejection in two cases at day 54 and day 88 and the histology of theirkidneys at autopsy demonstrated a dense lymphocytic infiltrate. In twoother cases, long-term unresponsiveness was observed (Table 7) to >127days and >79 days. The thymuses of the two monkeys which rejected theirgrafts were markedly decreased in size at autopsy compared toage-matched controls prior to treatment, but a small thymic remnant wasidentified.

The data demonstrate that anti-CD3-CRM9 is a potent, newimmunosuppressive agent which is capable of inducing tolerance inoutbred MHC class I and class II disparate rhesus monkeys. Thisattribute distinguishes it from other currently known immunosuppressiveagents, such as antithymocyte globulin, cyclosporine, or monoclonalantibodies which have more limited efficacy or safety in toleranceinduction in large mammals or which require more cumbersome strategies(Powelson et al., Transplantation 57: 788-793 (1994) and Kawai et al.,Transplantation 59: 256-262 (1995)). The degree of T cell depletionproduced by 3 doses of the drug is more complete than that achieved by alonger course of anti-lymphocyte globulin, which generally depletes to amuch lesser degree (Abouna et al., Transplantation 59: 1564-1568 (1995)and Bourdage J S, Hamlin D M, Transplantation 59:1194-1200 (1995)).Unlike OKT3, an activating antibody which does not necessarily kill Tlymphocytes, anti-CD3-CRM9 is a lytic therapy with a more profoundeffect on T cells than OKT3 and better potential for toleranceinduction. Its efficacy may be in part related to its ability to depleteT cells in the lymph node compartment, as well as in peripheral blood,since the majority of potentially alloreactive T cells reside in thelymph node compartments. The T cell. depletion produced by anti-CD3-CRM9is more complete than that achieved by any other known pharmacologicmeans, including total lymphoid irradiation, and it avoids the toxicside effects of radiation. Following treatment with the anti-CD3-CRM9,the thymus decreases markedly in size, although thymic cortex andmedullary structures are still apparent. Anti-CD3-CRM9 appears to besafe and well tolerated in rhesus monkeys. No significant adverse drugeffects were encountered. About half of the monkeys were treated withintravenous fluids for 3 to 5 days following administration to preventdehydration. No infections were encountered in these experiments andonly routine perioperative antibiotic prophylaxis was used at the timeof renal transplantation and thymic injection. Cytokine release syndromewas not seen and monkeys did not develop febrile illness following drugadministration.

The induction of tolerance in monkeys receiving thymic injection ofeither donor lymphocytes or normal saline in conjunction withanti-CD3-CRM9 suggests that thymic injection may provide an adjunct totolerance induction using T cell depletion with anti-CD3- CRM9.Presumably, CD3+lymphocytes present in the donor lymphocyte inoculum arealso killed by the drug administered to the recipients. This would leavedonor B cells to express donor MHC class I and class II in the recipientthymus. Rodent studies would suggest that it is the presence of one orboth of these antigens that is crucial to promoting thymic tolerance(Goss J A, Nakafusa Y, Flye M W, Ann Surg 217: 492-499 (1993); Knechtleet al., Transplantation 57: 990-996 (1994) and Oluwole et al.,Transplantation 56: 1523-1527 (1993)). Of even more interest is theobservation that normal saline injected into the thymus in conjunctionwith anti-CD3-CRM9 produced tolerance in two of two recipients.Surprisingly, the success of this approach suggests that immunotoxinrather than thymic injection is crucial. Alternately, non-specificdisruption of thymic integrity may contribute The observation that twoof four recipients treated with anti-CD3-CRM9 alone became tolerantsuggests that transient depletion of T cells by the drug is crucial inpromoting tolerance. In rodents, transplant tolerance can be achieved byconcomitant administration of donor antigen and anti-T-cell agents (QinS et al., J Exp Med 169: 779-794 (1989); Mayumi H, Good R. A., J Exp Med1989; 169: 213-238 (1989); and Wood M L et al., Transplantation 46:449-451 (1988)), but this report demonstrates donor-specific toleranceusing T cell specific therapy alone. The depletion of T cells from thelymph node compartment by anti-CD3-CRM9 may be crucial in promoting itsefficacy as a tolerance inducing agent and differentiate it fromanti-CD3 mAb alone which depletes the peripheral blood CD3 cells, buthas a weaker effect on the lymphoid tissues (Hirsch et al., J Immunol140: 3766-3772 (1988)).

These experiments using an outbred, MHC incompatible non-human primatemodel provide a rationale for tolerance strategies in human organtransplantation. The results are unique in offering a simple, reliable,and safe approach to tolerance in a model immunologically analogous tohuman solid organ transplantation. An anti-human CD3 immunotoxin (e.g.,scUCHT1-DT390 and anti-CD3-CRM9) has been constructed and has T cellkilling properties similar to FN18-CRM9 (see Neville 1992 and Neville1996). The preliminary results reported here have broad implications fortolerance in humans.

In summary, immunotoxin treatment alone leads to marked prolongation ofgraft survival in 100% of the cases to date. Eliminating the thymicmanipulation did not alter the success rate. No other drug or treatmentregimen comes close to achieving these results in primates.

TABLE 8 Skin Graft Results 3rd party Interval after skin survival Donorskin Monkey kidney transplant (days) survival (days) 93023 182 10 >36792108 140 1040 (and renal allograft rejection) POF 147 10 >221 POJ 18810 >152 PIP 176 10   >74

EXAMPLE 14 Immunotoxin Alone Induces Tolerance

Depletion of mature T cells can facilitate stable acceptance of MHCmismatched allografts, especially when combined with donor bone marrowinfusion. Although ATG and anti-T cell mAbs eliminate recirculatingcells, residual T cells in lymphoid tissue have potential to orchestrateimmune recovery and rejection. Unlike pure antibodies, CD3-immunotoxin(CD3-IT) can destroy cells following direct binding and intracellularuptake without limitations of immune effector mechanisms. Thus, CD3-ITmay have superior immunosuppressive activity. The action of CD3-IT inrhesus monkey kidney transplant recipients was examined.

The present example of CD3-IT is a conjugate of IgG1 mAb anti-rhesus CD3epsilon (FN18) and a mutant diphtheria toxin CRM9 (FN18-CRM9). The Bchain of CRM9 diphtheria toxin bears a mutation that markedly reducesbinding to diphtheria toxin receptors, allowing specificity to bedirected by anti-CD3.

CD3-IT was administered to 3-5 kg normal male rhesus monkey allograftrecipients at a dose of 67 μg/kg on days-1 and 33 μg/kg on days +0 and+1 without additional immunosuppressive drugs. Recipient-donorcombinations were selected to be incompatible by MLR and multiple DRallele mismatches; and all were seronegative for CRM9-reactive antibodyto diphtheria toxin. Three groups received CD3-IT: (1) alone (n=3), (2)in combination with day 0 infusion of donor bone marrow DR⁻CD3⁻ (n=3),(3) or with donor bone marrow and 200 cGy lymphoid irradiation given ondays −1 and 0 (n-3).

Kidney allograft survival was remarkably prolonged. With CD3-IT alone,graft survival time was 57, 51, and 44 days. In combination with donorbone marrow infusion, graft survival was >400, 124, and 36 days. CD3-IT,lymphoid irradiation, and donor bone marrow resulted in graft survivalof >300, 143, and 45 days. Both the 36 or 45 day graft losses were fromhydronephrosis without evidence of rejection. Peripheral blood T cellcounts fell selectively by 2 logs, and time to 50% recovery was 20-60days. The peripheral blood CD3+CD4/CD8 ratio increased 2-6 fold beforeadjusting to baseline by 3 weeks. B cell/T cell ratios in lymph nodeswere elevated >40-fold on day 5-7, reflecting a 1-2 log reduction incirculating and fixed tissue T cell compartments. LN CD4/CD8 ratios werenormal at 5-7 days, but CD45RA+CD4 and CD28-CD4 cell subsetsincreased >1 log while CD28+CD8 cells decreased by >1 log, suggestingfunctional subset changes.

Anti-donor MLR responses became reduced uniformly, but specificunresponsiveness was seen only in the donor bone marrow-treated group.Peripheral blood microchimerism was detectable by allele specific PCRafter donor bone marrow-infusion. These studies show CD3-IT to be anunusually effective and specific immunosuppressive agent in non-humanprimate transplantation and provides clinical tolerance inductionstrategies applicable to transplantation in humans.

EXAMPLE 15 Immunotoxin Plus Short Term Immunosuppressant Drugs InducesTolerance in Monkeys in Models Simulating Human Cadaveric Donors

The efficacy of IT in prolonging allograft survival was evaluated in amodel that stimulates transplantation of organs from cadaveric donors inhumans. Rhesus monkey donor-recipient pairs were selected on the basisof MHC class I and II disparity. Monkeys were given anti-CD3-CRM9immunotoxin 0.2 mg/kg iv in three divided daily doses starting on theday of the renal allograft (group 1). In group 2, recipients alsoreceived methylprednisolone 125 mg iv daily for 3 days and mycophenolatemofetil 250 mg po daily for 3 days starting on the day of thetransplant. Rejection was monitored by serum creatinine levels andconfirmed histologically.

Graft Survival (days) Group 1 Group 2 Group 3 (IT alone) (IT + MMF +methylprednisolone) (untreated) 79 >90 5 57 >75 7 51 >60 7 >124 >102

The short burst of intensive anti-T cell therapy given at the time ofthe transplant appears to be well tolerated and to reliably result inlong-term allograft survival. The mRNA cytokine profile of graftinfiltrating cells obtained from renal transplant biopsies in thisprotocol suggests that IL-2 and γ-IF (TH₁ associated) are present inmeasurable levels and IL-4 and 10 (TH₂ associated) are detected at muchlower levels. These results in a non-human primate model provide astrategy that can be applied to human organ transplant recipients whowould benefit substantially from independence from maintenanceimmunosuppressive drugs.

A second group of rhesus monkeys undergoing mismatched renaltransplantation received anti-CD3-CRM9 (IT) 18 hours pretransplant,0.067 mg/kg and 0.033 mg/kg on days 0 and +1. Group 1 received only IT,n=6. Group 2, n=7, received in addition to IT deoxyspergualin (DSG) IV2.5 mg/kg/day and solumedrol (SM), 7, 3.5 and 0.33 mg/kg IV during theIT administration. DSG was continued from 4 to up to 14 days. Plasmasamples were tested by ELISA for cytokine release syndrome by measuringpre and post transplant plasma IL-12 and INF gamma levels.

Graft Survival (days) Group 1 (IT alone) Group 2 (IT + DSG + SM) 10-57 n= 6 (rejections) >155-200 n = 4 28-45 n = 3 (rejections) 2 deaths fromnon-rejection causes

IT, Group I, (or rhesus anti-CD3 an antibody alone) elevated both IL-12and INF-8 gamma. DSG and solumedrol appear to block IL-12 inducedactivation of INF-gamma by a mechanism that may be associated withNF-kappa/beta (see FIGS. 20-21). This treatment is found to eliminateperitransplant weight gain (FIG. 22) and serum hypoproteinemia (FIG.23), both signs of vascular leak syndrome, which in this study isassociated with early graft rejection. This peritransplant treatmentregimen can provide a rejection-free window for tolerance inductionapplicable to cadaveric transplantation.

It takes over 24 hours for IT to exert most of its lymph node T cellkilling effects. Therefore, IT cadaveric transplantation protocols(protocols in which organ transplantation occurs generally within 6hours of initial therapy and not longer than 18 hours) benefitsubstantially from peritransplant supplemental short termimmunosuppressant agents to minimize peritransplant T cell responses tothe new organ as shown by the above data.

Throughout this application various publications are referenced bynumbers within parentheses. Full citations for these publications are asfollows. Also, some publications mentioned hereinabove are herebyincorporated in their entirety by reference. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart to which this invention pertains.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

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1. A method of inhibiting a rejection response in a primate recipient toforeign mammalian donor cells, tissue or organ, comprising the stops of:a) administering to the recipient an anti-CD3-DT immunotoxin, whereinthe immunotoxin comprises sFv-DT390, so as to reduce the recipient'sT-cell lymphocyte population by at least 80%, as compared to therecipient's T-cell lymphocyte population prior to administration of theimmunotoxin; and b) transplanting the donor cells, tissue or organ, intothe recipient, such that a rejection response by the recipient to thedonor cells, tissue or organ, is inhibited.
 2. The method of claim 1,wherein the donor cell constitute an organ.
 3. The method of claim 1,wherein the donor cells constitute tissue from an organ.
 4. The methodof claim 1, wherein the donor cells are allogeneic.
 5. The method ofclaim 1, wherein the donor cells are xenogeneic.
 6. The method of claim1, further comprising administering an immunosuppressant compound toenhance the anti-T cell effects of the immunotoxin.
 7. The method ofclaim 6, wherein the immunosuppressant compound is cyclosporin.
 8. Themethod of claim 6, wherein the immunosuppressant compound ismycophenolate mofetil.
 9. The method of claim 6, wherein theimmunosuppressant compound is deoxyspergualin.
 10. The method of claim6, wherein the immunosuppressant compound blocks IL-12-induced inductionof interferon-γ.
 11. The method of claim 6, wherein theimmunosuppressant is administered beginning from about 0 to 6 hoursbefore the transplanting step.
 12. A method of inhibiting a rejectionresponse in a primate recipient, by inducing immune tolerance to foreignmammalian donor cells, tissue or organ, comprising the steps of: a)administering to the recipient an anti-CD3-DT immunotoxin, wherein theimmunotoxin comprises sFv-DT390, so as to reduce the recipients's T-celllymphocyte population by at least 80%, as compared to the recipient'sT-cell lymphocyte population prior to administration of the immunotoxin;b) administering an immunosuppressant compound to enhance the anti-Tcell effects of the immunotoxin; and c) transplanting the donor cells,tissue or organ, into the recipient, such that a rejection response bythe recipient to the donor cells, tissue or organ, is inhibited.
 13. Themethod of claim 12, wherein the immunosuppressant compound isdeoxyspergualin.
 14. The method of claim 12, wherein theimmunosuppressant compound blocks IL-12-induced induction ofinterferon-γ.
 15. The method of claim 12, wherein the immunosuppressantis administered beginning from about 0 to 6 hours before thetransplanting step.